doi:10.1093/brain/awu205

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BRAIN A JOURNAL OF NEUROLOGY

Intrathecal somatic hypermutation of IgM in multiple sclerosis and neuroinflammation Eduardo Beltra´n,1,2 Birgit Obermeier,1 Markus Moser,3 Francisco Coret,4 Marı´a Simo´-Castello´,2 Isabel Bosca´,2 Francisco Pe´rez-Miralles,2 Luisa M. Villar,5 Makbule Senel,6 Hayrettin Tumani,6 Reinhard Hohlfeld,1,7 Bonaventura Casanova2 and Klaus Dornmair1,7 1 2 3 4 5 6 7

Institute of Clinical Neuroimmunology, University Hospital Grosshadern, LMU Munich, D-81377 Munich, Germany Unidad Mixta de Esclerosis Mu´ltiple y Neurorregeneracio´n, IIS Hospital La Fe — Universitat de Vale`ncia, 46026 Valencia, Spain Department for Molecular Medicine, Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany Neurology Department, Clinic University Hospital, 46026 Valencia, Spain Department of Immunology, Hospital Ramo´n y Cajal, 28034 Madrid, Spain Department of Neurology University of Ulm, D-89081 Ulm, Germany Munich Cluster for Systems Neurology (SyNergy), Adolf-Butenandt-Institut, Ludwig-Maximilians-Universita¨t Mu¨nchen, 80336 Mu¨nchen, Germany

Intrathecal oligoclonal bands of the cerebrospinal fluid are considered the most important immunological biomarkers of multiple sclerosis. They typically consist of clonally expanded IgG antibodies that underwent affinity maturation during sustained stimulation by largely unknown antigens. In addition, 40% of patients with multiple sclerosis have oligoclonal bands that consist of expanded IgM antibodies. We investigated the molecular composition of IgM- and IgG-chains from cerebrospinal fluid of 12 patients with multiple sclerosis, seven patients with other neurological diseases, and eight healthy control subjects by highthroughput deep-sequencing and single-cell PCR. Further, we studied the expression of activation-induced cytidine deaminase, the key enzyme for affinity maturation of antibodies, in cerebrospinal fluid samples of 16 patients. From the cerebrospinal fluid of two multiple sclerosis patients we isolated single B cells and investigated the co-expression of antibody chains with activation-induced cytidine deaminase. In striking contrast to IgM-chains from peripheral blood, IgM-chains from cerebrospinal fluid of patients with multiple sclerosis or neuroborreliosis showed a high degree of somatic hypermutation. We found a high content of mutations that caused amino acid exchanges as compared to silent mutations. In addition, more mutations were found in the complementarity determining regions of the IgM-chains, which interact with yet unknown antigens, as compared to framework regions. Both observations provide evidence for antigen-driven affinity maturation. Furthermore, single B cells from the cerebrospinal fluid of patients with multiple sclerosis co-expressed somatically hypermutated IgM-chains and activation-induced cytidine deaminase, an enzyme that is crucial for somatic hypermutation and class switch recombination of antibodies and is normally expressed during activation of B cells in germinal centres. Clonal tracking of particular IgM + B cells allowed us to relate unmutated ancestor clones in blood to hypermutated offspring clones in CSF. Unexpectedly, however, we found no evidence for intrathecal isotype switching from IgM to IgG. Our data suggest that the intrathecal milieu sustains a germinal centre-like reaction with clonal expansion and extensive accumulation of somatic hypermutation in IgM-producing B cells.

Keywords: multiple sclerosis; neuroborreliosis; oligoclonal bands; cerebrospinal fluid; immunoglobulin Received February 3, 2014. Revised June 16, 2014. Accepted June 24, 2014. Advance Access publication July 23, 2014 ß The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]

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Correspondence to: Klaus Dornmair, PhD, Institute of Clinical Neuroimmunology, University Hospital Grosshadern, LMU Munich, D-81377 Munich, Germany E-mail: [email protected]

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Abbreviations: C = constant; CDR = complementarity determining regions; FR = framework region; Ig = immunoglobulin; SHM = somatic hypermutation; V = variable; VH = variable heavy

Introduction

Materials and methods

Clinical samples The clinical data of the patients are listed in Table 1. We investigated 12 patients with multiple sclerosis (Patients MSM-1 to MSM-12), two patients with neuroborreliosis (Patients NBM-1 and NBM-2), one patient with hyperacute disseminated encephalomyelitis-like disease (Patient ADEM), one patient with neuromyelitis optica (Patient NMO), one patient with facial palsy (Patient FP), and two patients with chronic adult hydrocephalus dementia (Patients CHD-1 and CHD-2). All patients with multiple sclerosis had a relapsing-remitting disease course. All patients were untreated at the time of lumbar puncture, except Patients MSM-2, MSM-3, and MSM-5 who received natalizumab. Paired serum and CSF samples were stored at 80 C in aliquots. For determination of IgG and IgM indices, levels of IgG, IgM, and albumin were quantified in paired serum and CSF samples using a Siemens nephelometer. IgG and IgM oligoclonal band detection was performed blinded to clinical and MRI data by isoelectric focusing before immunodetection as described (Villar et al., 2001; Sadaba et al., 2004). CSF cells from patients were processed and managed by the Biobank La Fe, Valencia, Spain, following legal and ethical requirements. Samples from patients with neuroborreliosis were collected and processed at the Department of Neurology, University of Ulm, Germany. For single cell analysis, CSF cells were stored alive in foetal bovine serum containing 10 % dimethylsulphoxide in liquid nitrogen.

Generation of Ig transcript data sets Fresh CSF samples (3–5 ml) were centrifuged at 1000g for 10 min. Peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation. RNA was isolated using NucliSens easyMag automated extractor (bioMe´rieux) or by phenol extraction (Obermeier et al., 2008). Reverse transcription and two rounds of PCR amplification were carried out as described (Obermeier et al., 2008). Outer (suffix ‘out’) and inner (suffix ‘in’) primers were used at 0.5 mM per reaction in two independent rounds of nested PCR. In addition, IgM-specific reverse primers (suffix ‘Cmu’) (Supplementary Table 1) were included. Further, all reverse inner (‘rev-in’) primers were tagged with multiplex identifier for sample identification. Nested multiplex PCR products for IgM and IgG obtained from each sample were purified by QIAquickÕ Gel Extraction Kit (QIAGEN) and pooled in equimolar concentrations. The mixtures were further processed by the Pyrosequencing Service in the Center for Public Health Research, Valencia, Spain, where adaptors A and B were ligated and the samples sequenced with a Roche GS FLX sequencer and Titanium chemistry.

Study approval

Sequence analysis and construction of lineage trees

The study was approved by the ethics Committees of the University Hospital La Fe, the Clinic University Hospital of Valencia, Spain, and

A total of 12 459 productively rearranged sequences were analysed using the programs IMGT/HighV-QUEST (Larkin et al., 2007) tool.

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Intrathecal antibody production is a hallmark of inflammatory diseases of the CNS. It is observed in diseases with presumed autoimmune pathogenesis such as multiple sclerosis, and also in infectious diseases such as neuroborreliosis (Meinl et al., 2006; Awad et al., 2010; von Budingen et al., 2011; Stangel et al., 2013). Typically, patient-specific patterns of expanded immunoglobulin (Ig) populations may be visualized by isoelectric focusing as oligoclonal bands. Oligoclonal bands are produced by CSF-resident B cells, the repertoires of CNS- and CSF-resident B cells overlap, identical B cell clones may be found at different locations in the brain, and B cells may migrate across the blood–brain barrier in both directions (Obermeier et al., 2008, 2011; Lovato et al., 2011; von Budingen et al., 2012). Most oligoclonal bands are of the IgG isotype, i.e. they have undergone class switch recombination and somatic hypermutation (SHM) in germinal centres (Seifert and Kuppers, 2009). Both processes are regulated by activation-induced cytidine deaminase (AICDA) (Xu et al., 2012; Robbiani and Nussenzweig, 2013), an enzyme expressed as several splice variants in activated B cells during the germinal centre reaction (Wu et al., 2008). However, AICDA is not expressed in resting B cells, and it is rapidly downregulated after B cell activation. The properties of IgG oligoclonal bands suggest an antigen-driven immune response, but the target antigens are so far largely unknown. In addition to IgG oligoclonal bands, persisting oligoclonal bands of the IgM isotype can be identified in the CSF of 40% of patients with multiple sclerosis (Villar et al., 2005; Thangarajh et al., 2008). This is surprising as IgM antibodies are usually naı¨ve, germlineencoded Ig which are produced by B cells that have not yet encountered antigens. To gain a better understanding of the IgM repertoire of CSF-resident B cells, we applied high-throughput sequencing and single cell analysis. In striking contrast to the IgM repertoire of B cells in blood, we found that the intrathecal IgM repertoire is characterized by a high degree of SHM. Clonal tracking allowed us to construct clonal lineage trees relating IgM + precursor clones in blood to hypermutated offspring clones in CSF. Moreover, we could detect AICDA transcripts in single B cells from CSF of patients with multiple sclerosis in conjunction with somatically mutated IgM- and IgG-chains. Our findings reveal a novel, unexpected property of the intrathecal IgM repertoire in neuroinflammatory diseases. The intrathecal milieu appears to sustain an unusual type of IgM response that is characterized by AICDA expression, proliferation, and presumably antigen-driven extensive accumulation of SHM.

the University of Ulm, Germany. Multiple sclerosis patients were diagnosed of relapsing-remitting multiple sclerosis according to the 2005 McDonald criteria (Polman et al., 2005). Informed consent was obtained from all patients and healthy control subjects according to the Declaration of Helsinki.

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Table 1 Clinical data of patients Patient

Age Sex Follow-up Relapses EDSS Treatment

MSM-1 MSM-2 MSM-3 MSM-4 MSM-5 MSM-6 MSM-7 MSM-8 MSM-9 MSM-10 MSM-11 MSM-12 ADEM NMO FP NBM-1 NBM-2 CHD-1 CHD-2

25 29 39 19 32 39 26 41 60 40 46 33 39 43 33 25 75 79 85

F F M F F M F M M F F F M F F M F F M

33 48 18 12 43 18 123 99 51 27 49 22 13 26 2

2 2 2 1 8 3 4 1 2 1 3 4 1 1 1

1.0 2.5 4.5 2.5 2.0 2.0 6.5 3.5 7.5 2.0 4.0 1.0 4.0 1.5 2.0

IgG-I IgM-I IgG-OCB IgM-OCB IgG PCR IgM PCR Pyroseq. AICDA

0.60 Natalizumab 1.49 Natalizumab 0.82 2.41 Natalizumab 0.86 3.26 0.55 0.75 0.74 0.60 0.97 1.28 0.45 1.96 0.76 0.6 2.45 0.42 0.43

0.18 1.11 0.07 0.48 0.10 0.10 0.04 0.18 0.08 0.05 0.08 0.10 0.06 0.06 0.05 0.79 4.26 0.05 0.05

+ + + + + + + + + + + + Neg + + + + Neg Neg

+ + + + + + + + + + + +* + + + + + Neg Neg

+ + + + + + + + + + + scPCR + scPCR Neg + Neg + + Neg Neg

+ + + + + + + + + + + scPCR + scPCR + + + + Neg Neg Neg

yes yes yes yes yes yes yes yes yes yes n/a n/a yes n/a n/a yes yes n/a n/a

Neg
E4a FL,
E4 Neg
E4a
E4 Neg
E4,
E4a n/a Neg FL,
E4,
E4a FL,
E4a Neg FL,
E4 Neg n/a n/a Neg Neg

Only sequences longer than 300 nucleotides that covered the antibody variable domains and contained 424 nucleotides of the conserved region were considered to unambiguously discriminate IgG and IgM chains. IgG and IgM variable heavy (VH) sequences of each patient were aligned with their corresponding VH germline sequences, thus enabling us to determine the rates of SHM in the complementarity determining regions (CDR) 1 and 2 compared to those within the framework regions (FR) 1, 2 and 3. CDR3 and FR4 were not included in the SHM analysis. However, CDR3-FR4 amino acid sequences were used to cluster sequences which belong to clonally related lineages. Alignment of the sequences was generated using ClustalW2 (Larkin et al., 2007). Rooted phylogenetic trees with branch lengths were generated by UPGMA (Unweighted Pair Group Method with Arithmetic mean) where sequences are clustered based on sequence similarity and branch length is equivalent to the number of changes that have occurred. Only those sequences sharing identical CDR3-FR4 were used to generate lineage trees using the IgTree software (Barak et al., 2008), which was kindly provided by Prof. Dr Ramit Mehr, Ramat-Gan, Israel. Lineage trees were displayed using the hierarchic layout in Cytoscape-2.8.3 (Smoot et al., 2011).

Clonal tracking in peripheral blood To relate clones that were expanded in the CSF to precursors in peripheral blood, we used cDNA from peripheral blood (see above) as template for clone-specific PCR. We amplified IgG- and IgM-heavy chains by two independent rounds of PCR. First, a nested PCR was carried out as described (Obermeier et al., 2008). Then a second nested PCR was performed in individual reactions using clone-specific primers. To this end, we used clone-specific forward primers that

hybridized upstream of the CDR1 region so that the 3’-nucleotides of the inner primers matched to nucleotides introduced by SHM. In addition we used primers matching to germline sequences to identify parent germline encoded chains. The reverse primers were designed so that their 5’-ends matched to N-nucleotides of the CDR3 regions. All clone-specific primers are listed in Supplementary Table 1.

Single cell analysis CSF cells stored in liquid nitrogen were thawed, B cells isolated using the BCLL B-Cell Isolation Kit (Miltenyi), and single B cells sorted into 96 well plates using a BD FACSAriaTM 2 flow cytometer. Nested multiplex PCR for IgM, IgG and AICDA was performed using the QIAGEN One-Step RT-PCR Kit. The conditions were as described by Obermeier et al. (2008), with minor modifications: we used gene-specific primers for cDNA synthesis (‘RT primers’) at 0.2 mM each in the reaction mix. After cDNA synthesis, a mixture of all outer primers was added to each well (0.2 mM of each primer in a final reaction volume of 50 ml) and PCR was performed as described (Obermeier et al., 2008), with an added heat activation step of 95 C for 10 min. A second nested PCR for each gene was performed in independent reactions using gene specific primers. Nested PCRs were carried out as described (Obermeier et al., 2008). Primers used in the single-cell (‘sc’) analysis are listed in Supplementary Table 1.

Statistics All statistical analyses were computed with GraphPad Prism software. Significant differences between groups were assessed using Tukey’s test. P-values 50.05 were considered statistically significant.

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We list the patients, their age and sex (F, female; M, male), the time of follow-up in months, number of relapses since disease onset (Relapses), their expanded disability status scale (EDSS), treatment at the time of the lumbar puncture, the IgG index (IgG-I), the IgM index (IgM-I), and the presence or not of both CSF specific oligoclonal IgG bands (IgG-OCB) and CSF specific oligoclonal IgM bands (IgM-OCB). In the last four columns we list whether or not we obtained PCR products for IgG and IgM, whether PCR products were subjected to pyrosequencing, and which AICDA isoform was found by conventional cloning and sequencing. All patients with multiple sclerosis (MSM) had a relapsing-remitting disease course. We did not obtain PCR products for IgG- and IgM-heavy chains from the two patients with chronic adult hydrocephalus dementia (CHD), no IgM PCR product from Patient NBM-2, and no IgG PCR product both from Patients ADEM and BPM. + = positive; Neg = Negative; n/a = not available; scPCR, single-cell PCR analysis. *Patient MSM-12 showed IgM oligoclonal bands in CSF and serum. ADEM = hyperacute disseminated encephalomyelitis-like disease; NBM = neuroborreliosis; FP = facial palsy.

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acid sequences were deduced from nucleotide sequences which were obtained by pyrosequencing of B cells. The 12 most frequently found sequences are listed for: (A) IgG-chains from blood; (B) IgM-chains from blood; (C) IgG-chains from CSF; and (D) IgM-chains from CSF. In the uppermost line, the putative positions of the framework region and CDR are indicated. The first column denotes how often a particular sequence was found. The second column lists the germline V families and alleles of the chains. The third column lists the deduced amino acid sequences of the Ig-heavy chains. Germline-encoded amino acids are shown in black letters. Amino acids that were introduced by SHM or comprise the NDN nucleotides of the CDR3 are shown in red letters. The sequences are aligned to the conserved cysteine residues of the V regions, the tryptophan glycine residues of the joining (J) regions and the C region residues, which are indicated by grey backgrounds. In the fourth column, we indicate with the same number of asterisks (*) the sequences sharing identical CDR3-FR4 regions. All IgM sequences from CSF of patient MSM-1 are shown in Supplementary Fig. 1.

Results Repertoire analysis of IgM and IgG chains from blood and CSF We investigated the IgG- and IgM-heavy chain transcript sequences from CSF-resident B cells of 10 patients with multiple sclerosis known to be positive for IgG and IgM oligoclonal bands (Patients MSM-1 to MSM-10) by high-throughput pyrosequencing. In addition, we analysed CSF from two patients with neuroborreliosis (Patients NBM-1 and NBM-2), one patient with hyperacute disseminated encephalomyelitis-like disease (Patient ADEM), one patient with neuromyelitis optica (Patient NMO), one patient with facial palsy (Patient FP), and two patients with chronic adult hydrocephalus dementia (Patients CHD-1 and CHD2). Further, we analysed IgG and IgM transcripts of blood from four patients with multiple sclerosis (Patients MSM-1, -2, -3, and -9) (Table 1), and from eight healthy control subjects (HC-1 to HC-8). The clinical data of the patients are listed in Table 1. We

used pyrosequencing, a second-generation sequencing technique that may yield reads of 5300 bp (Metzker, 2010), which allowed us to analyse Ig sequences that span the entire variable (V) Ig region including at least 24 nucleotides of the constant (C) regions to allow for definite discrimination of IgM and IgG. We obtained 12 459 productively rearranged sequences of IgG- and IgM-heavy chains with 4506 of them being unique at the amino acid level (Supplementary Table 2). The deduced amino acid sequences were aligned according to their VH germline sequences. As an example, we show the most frequently found IgM and IgG chains from CSF and blood of Patient MSM-1 in Fig. 1, and the complete set of all IgM sequences from CSF in Supplementary Fig. 1. In particular in CSF samples, many sequences were found several times in the same patient, as expected for clonally expanded populations. The amino acids introduced by SHM are highlighted in red letters in Fig. 1. They are detected throughout the V-regions. As expected, many amino acids were altered by SHM in IgG-chains from blood and CSF (Fig. 1A and C). Few altered amino acids were observed in IgM-chains from blood (Fig. 1B). Again, this is expected as it is

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Figure 1 Alignment of amino acid sequences of IgM- and IgG-chains from CSF and blood of multiple sclerosis Patient MSM-1. Amino

Somatic hypermutation of intrathecal IgM

Clonal relationship between blood and CSF B cells To investigate whether B cell clones from CSF are related to B cell clones from peripheral blood, we tried to amplify VH transcripts of particular expanded CSF clones in cDNA samples from blood using clone-specific PCR primers (see ‘Materials and methods’ section). Thereby we focused on chains that belonged to the same VH family and had identical CDR3 sequences. In the VH4-59 family of Patient MSM1 we identified related clones when we tracked IgM chains from CSF in the IgM compartment of blood (Fig. 4A). In the same patient we could also identify related IgG VH3-7 clones from CSF and blood (Fig. 4B). In both families we also found chains that had identical nucleotide sequences in addition to several other clones where only few nucleotides were

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exchanged by SHM. Of note, in the IgM VH4-59 family we could identify the parent germline sequence of an expanded CSF clone in blood. Interestingly, we did not find any related IgM chain in the IgG compartment and vice versa, indicating that the different families developed independently. In Patient MSM-2, we found comparable results in the VH5-51 family (Supplementary Fig. 3). Here we could identify six clones that had identical amino acid sequences, which were encoded by different nucleotides. This suggests that clonal development was triggered by particular antigen(s).

Nucleotide and amino acid replacements introduced by somatic hypermutations in CSF-resident B cells To quantify the extent of SHM in CSF and blood of patients and controls, we determined the SHM frequencies SHM, i.e. the numbers of mutated nucleotides per chain length for all IgM- and IgG-chains. Because SHM is typically found in all IgG-chains, we normalized the SHM frequencies of IgM-chains SHM-IgM to the SHM frequencies of the corresponding IgG-chains SHM-IgG. The quotients SHM-IgM/SHM-IgG are a measure for IgM-specific SHM. They are equal in blood from patients and controls (0.38 and 0.48, respectively), whereas they were elevated up to 0.88 in CSF from patients (Fig. 5A). This shows that considerable SHM occurs in IgM-chains from the CSF of patients with multiple sclerosis, but not in peripheral blood of patients or controls. Under normal conditions, SHM is driven by antigens in mature B cells in germinal centres (Xu et al., 2012; Robbiani and Nussenzweig, 2013). Therefore SHM is higher in the CDR which interact with antigens as compared to the framework regions that do not contact antigens. Further, in antigen-experienced B cells, the frequency of nucleotide replacements leading to amino acid exchanges (‘replacement mutations’) SHM-R is higher than the frequency of substitutions not leading to amino acid exchanges (‘silent mutations’) SHM-S due to positive selection of clones that interact with antigens. Therefore we analysed the SHM frequencies SHM separately in the framework region and CDR of IgGand IgM-chains at the nucleotide level (Fig. 5B). Further, we compared the frequency ratios of replacement and silent mutations SHM-R/SHM-S independently in the framework region and CDR (Fig. 5C). Figure 5B shows that SHM of IgG-chains in CSF and in blood of patients with multiple sclerosis and those with neuroborreliosis were significantly higher in the CDR than in the framework region. By comparison, SHM of IgM-chains were approximately identical in CDR and the framework region in blood of patients and healthy control subjects. However, they were strikingly higher in the CDR than in the framework region of IgM-chains from the CSF of patients as compared to healthy control subjects. They reach levels comparable to IgG-chains, suggesting that in CSF also the IgM responses might be driven by sustained exposure to antigens. Figure 5C shows the R/S ratio SHM-R/SHM-S for IgG- and IgM-chains from CSF and blood of patients and healthy control subjects. An R/S ratio of 2.9 is known to be typical for random mutations in an Ig region before contact with antigens (Jukes and King, 1979). An R/S

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known that only a minor fraction of IgM-chains from blood contains somatically hypermutated amino acids which are introduced in germinal centres (Seifert and Kuppers, 2009). For comparison, we show comparable sequences of IgM-chains from blood of control subject HC-1 in Supplementary Fig. 2. As expected, many mutations are found in IgG-, but not in IgM-chains. Comparable data sets were generated from all other patients and controls. Surprisingly, however, in IgM-chains from CSF (Fig. 1D) we observed about as many amino acids altered by SHM as observed in IgG-chains. Many IgM-chains shared identical hypervariable CDR3 and differed only in few amino acids in the V-regions. Sequence alignment showed clusters of clonally related chains (Fig. 2), which clearly demonstrates the genealogy of affinity maturation. In Fig. 2A we show the alignment of all IgM-chains form the CSF of Patient MSM-1, as listed in Supplementary Fig. 1. Chains were grouped by their VH-family and are distinguished by different colours. In Figs. 2B and C we analysed the clonal genealogy of the VH-18*01 family (Fig. 2A) and of the VH4-59 family (Fig. 2A) using the program IgTree. For comparison, we show an identical analysis of CSF IgG-chains in Fig. 3. Figure 3A shows the analysis of all detected IgG-chains and Figs 3B and C show exemplarily the genealogies of the VH-18*01 and VH37*01 families. This detailed analysis illustrates how the different chains interrelate to each other and reveals their common ancestors. The structures and genealogies of IgM and IgG families were comparable. Of note, we found a preponderance of the VH4 family not only in IgG-chains from several patients (Patient MSM-1: VH4-34 and 4-39; Patient MSM-2: VH4-59; Patient MSM-3: VH4-4 and 4-59; Patient MSM-5: VH4-34 and 4-59; Patient MSM-8: VH4-59) but also in IgM-chains [Patient MSM1: VH4-59 (Fig. 2C); Patient MSM-2: VH4-59 and 4-61; Patient MSM-5: VH4-31; Patient MSM-7: VH4-61; Patient MSM-10: VH4-31; Patient ADEM: VH4-4 and 4-39]. A VH4 bias in CSFIgG was found earlier by other groups (Owens et al., 1998, 2003, 2007; Qin et al., 1998; Baranzini et al., 1999; Obermeier et al., 2008; von Budingen et al., 2012), using PCR primers hybridizing to different sites within the VH cDNA. Of note, Owens et al. (1998) and von Budingen et al. (2012) used anchor-PCR, where all VH families are amplified by identical primers. Therefore a PCRbased bias may be excluded.

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Figure 2 Ig lineage tree constructed from IgM-VH sequences taken from CSF of Patient MSM-1. (A) Automated alignment was performed using ClustalW2 and rooted phylogenetic trees with branch lengths were generated by UPGMA software (see ‘Materials and methods’ section), where sequences are clustered based on sequence similarity, and branch length is equivalent to the number of changes that have occurred. First, sequences were clustered in three main clusters corresponding to each VH family (VH1, VH3 and VH4). Clusters with sequences sharing identical CDR3-FR4 regions were shaded with different colours, and are indicated by labels with the VH-family name. Amino acid sequences are listed in Supplementary Fig. 1. (B) Nucleotide sequences corresponding to the largest cluster represented in the upper panel (VH1-18) were used to generate a linage tree using IgTree software. In the Ig lineage tree, all single mutations separating the sequences are represented as individual round nodes. The putative germline sequence is labelled black. Each blue node represents at least one unique IgM-VH sequence; larger nodes represent up to hundreds of identical sequences. Hypothetical intermediates calculated by IgTree are labelled beige. Branched labels represent the number of mutational steps between nodes; only mutational steps 41 are indicated; thus, unlabelled branches represent a single mutation step. (C) Analysis of the phylogenetic tree of the IgM family VH4-59. See legend to Fig. 2B for details.

ratio exceeding 2.9 is therefore another strong indicator of antigen-driven affinity maturation. In all framework regions we indeed found values that were close to 2.9. Further, as expected, the CDR of IgG-chains from blood of patients and healthy control subjects showed elevated levels well above 2.9. Strikingly, we observed the highest R/S ratios for CDR of IgM and IgG from CSF from patients with multiple sclerosis with values of 6.6 and 6.2, respectively. This supports the interpretation above that an

unusual antigen-driven SHM of IgM might occur in the CSF of patients with multiple sclerosis.

AICDA expression in CSF B cells Because CSR and SHM are both regulated by the key enzyme AICDA, we tested whether CSF-resident B cells of patients with multiple sclerosis expressed AICDA transcripts. Using PCR primers that may amplify all AICDA isoforms (Wu et al., 2008), we found

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Figure 3 Multiple alignment of IgG-sequences from the CSF of Patient MSM-1. IgG families were analysed identically to the analysis of IgM families shown in Fig. 2. (A) Automated alignments were performed using ClustalW2. Phylogenetic trees of nucleotide sequences corresponding to the IgG VH1-18 family (B) and to the IgG VH3-7 family (C) as calculated by IgTree. See legend to Fig. 2 for details.

that the full-length AICDA (AICDA-FL) was only expressed in cells from Patient MSM-3, the splice variant AICDA-
E4 was expressed in Patients MSM-3, -6, and -8, and splice variant AICDA-
E4a was found in Patients MSM-2, -5, and -8 (Table 1). We could not detect AICDA expression in Patients MSM-1, -4, -7, and -10, which may be due to insufficient sensitivity of our assay or to the fact that AICDA is only expressed during the activation states of B cells (Xu et al., 2012; Robbiani and Nussenzweig, 2013). In two patients, MSM-11 and MSM-12, we analysed single B cells isolated by flow cytometry from CSF by single-cell multiplex PCR to investigate the expression of AICDA in conjunction with

IgM- and IgG-heavy chains. In both patients we detected IgMand IgG-chains with considerable extents of SHM whereas almost no germline sequences were found. However, we found many germline sequences by a parallel analysis of blood from a healthy control. In Patients MSM-11 and MSM-12 we detected several identical chains in different cells and found a number of closely related chains that differed only in one or few amino acids, many of them having identical CDR3 regions (Supplementary Figs 4 and 5). This confirms clonal IgM expansions in CSF-resident B cells as detected by pyrosequencing (Fig. 1) at the level of single cell analysis. In many cells, either IgG- or IgM-chains were co-expressed with AICDA. Exemplarily we show the results of five

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E. Beltra´n et al. variant. However, it is known that all splice variants contain exon 2, which induces SHM (Wu et al., 2008).

Discussion

Figure 4 Tracking of expanded CSF clones in peripheral blood.

single cells (sc) from MSM-11 in Fig. 6. In single cells sc1 and sc2 we detected an IgG-chain together with AICDA-FL; in sc3 we found IgM and AICDA-
E4a, and in sc4 IgM with AICDA-FL. In some cells we detected two different AICDA isoforms together with one IgM- or IgG-chain (Fig. 6, sc5). Although the molecular reasons are unknown, expression of two AICDA isoforms in a single cell was observed earlier (Wu et al., 2008). We could not detect Ig-chains and AICDA transcripts in all 475 cells that we analysed. This may be due to the limited sensitivity of the single-cell multiplex PCR and/or to partly degraded mRNA in the cells, which were frozen in foetal bovine serum and dimethylsulphoxide before purification by magnetic beads and single-cell flow cytometry. We could not detect an evident correlation between IgM or IgG expression and any particular AICDA splice

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Detailed analysis of clonal relations between expanded clones from CSF and clones from peripheral blood of Patient MSM-1. All clones belong to identical VH-families and share the same CDR3 sequences. Nucleotide sequences were used to calculate phylogenetic trees using IgTree software. (A) Analysis of the VH4-59 family. Blue nodes represent clones from CSF. They are identical to clones shown in Fig. 2C. Red nodes represent clones that were found in peripheral blood. Identical sequences in CSF and blood are depicted in green. Beige nodes represent clones that were calculated but not identified experimentally. Nodes depicted as squares indicate sequences that were identified by family PCR primers but may not be detected by clone-specific primers which hybridize downstream of the positions of the primers used for family PCR. (B) Analysis of the IgG VH3-7 family of Patient MSM-1. The colour code is identical to Fig. 4A. The black dot represents the parent germline VH3-7 clone which was not found experimentally. We note that we may exclude PCR contaminations because most sequences are slightly different.

We identified two important novel features of the intrathecal IgM antibody response: firstly, intrathecally produced IgM antibodies show extensive SHM, and secondly, intrathecal IgM-producing B cells express AICDA, the key enzyme supporting SHM in classical germinal centres. An inflammatory milieu likely triggers these unexpected features, because somatically mutated intrathecal IgMs are not specific for multiple sclerosis as we also found them in cases of neuroborreliosis, facial palsy, and neuromyelitis optica, i.e. in cases with neuroinflammation. In multiple sclerosis, IgM expression is observed only in a proportion of patients, but interestingly, patients with IgM oligoclonal bands were reported to have particularly severe disease courses (Villar et al., 2005, 2014; Thangarajh et al., 2008; Beltran et al., 2012). This also holds true for the patient cohort studied here (Table 1). The reasons are still unclear but may be related to yet unknown properties of the intrathecal milieu or to the nature of the still unknown target antigens. Some somatically hypermutated IgM-chains were highly expanded, and many chains were clonally related. These features strongly point to local activation and proliferation of IgM-producing B cells. This is consistent with an antigen-driven intrathecal process, because SHM occurred predominantly in the CDR where disproportionately many nucleotide exchanges led to amino acid substitutions. If this was a random process, the SHM rates would be equal in the framework region and CDR with average rates of replacement mutations. Because under normal conditions all AICDA isoforms are expressed exclusively in germinal centres, meningeal follicle–like structures (Serafini et al., 2004) are one of the candidate site(s) where intrathecal IgM-producing B cells might recognize antigens and become activated to express AICDA, proliferate, and undergo SHM. The presence of such AICDA-positive ectopic germinal centre-like structures was already detected in chronically inflamed tissues in several autoimmune disorders, such as rheumatoid arthritis (Humby et al., 2009), myasthenia gravis (Zuckerman et al., 2010), Sjo¨gren’s syndrome (Stott et al., 1998; Bombardieri et al., 2007), and systemic lupus erythematosus (Nacionales et al., 2009). Comparison of IgG and IgM sequences from CSF to sequences from blood reveals that the extent of SHM in IgG chains is similar. By contrast, IgM chains from CSF show a very high level of SHM whereas IgM chains from blood are predominantly germline with only few chains showing some mutations. It is known from classical studies (Klein et al., 1997) that SHM occurs only in a subset of peripheral IgM producing B cells and that most IgM chains in the periphery have germline sequences. Our observation that hypermutated IgM sequences are highly enriched in the CSF may reflect the special cellular composition of the CSF that is known to be enriched in CD27 + memory B cells (Corcione et al., 2004; Cepok et al., 2005). To further address this point it will be necessary to compare sorted subsets of B cells from blood and CSF. Another limitation is that in the current study we could

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Figure 5 Continued

Figure 5 Analysis of nucleotide and amino acid replacements by SHM in Ig-chains of patients and controls. (A) Quotients of SHM frequencies in IgM- and IgG-chains SHM-IgM/SHM-IgG. We determined the nucleotide exchange frequencies normalized to chain lengths by counting the nucleotides that differed from germline sequences. We compared CSF from 10 multiple sclerosis patients, peripheral blood samples from four patients with multiple sclerosis, and from eight healthy control subjects. Blood was

only available from 4 of 10 patients with multiple sclerosis. Blood samples from Patients NBM-1, NBM-2, ADEM, NMO, and FP were not available. Bars indicate standard deviation (SD). (B) Frequencies of nucleotide exchanges SHM for IgM- and IgGchains from CSF and blood from patients and healthy control subjects. The frequencies were calculated independently for CDR (red) and framework region (blue). We considered CDR1, CDR2, and FR1 to FR3, but omitted CDR3 and FR4. The data points are shown as circles for IgM-chains, and as squares for IgG-chains for each individual patient and healthy control subject. For each group of data the SD is shown as bar. *P 5 0.05; **P 5 0.01; # P 5 0.001. (C) Ratio of replacement to silent nucleotide exchanges SHM-R/SHM-S in CDR and framework region regions of patients and healthy control subjects in CSF and blood. The R/S quotient SHM-R/SHM-S which reflect the rates of nucleotide exchanges that lead to altered (R) or unchanged (S) amino acids were determined for all patients with multiple sclerosis. The R/S value of 2.9 is indicated by grid line. It indicates the rates of random mutations in an Ig region not under functional constraints on the protein level. IgM was only detected in one neuroborreliosis patient where SHM-R/SHM-S was slightly increased to 4.0. Of note, for unknown reasons the patient ADEM shows a dramatically increased SHM-R/SHM-S ratio of 65.0.

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perform the demanding single-cell PCR experiments only on a small number of cells. Nevertheless, our data clearly demonstrate co-expression of AICDA and IgM. However, we cannot rule out that some IgM + cells were already AICDA-positive when they entered the CSF from blood. Regardless of this possibility, our clonal tracking experiments formally demonstrate that IgM + B cells undergo massive clonal expansion and SHM in the CSF compartment. Regarding the properties of the IgM producing CSF-resident B cells analysed here, two observations may be relevant: firstly, we did not find any clonal overlaps between the IgG and the IgM CSF repertoires. In fact, we did not even find a single CDR1 or CDR2 that was shared between an IgG and an IgM-chain of a particular patient. This surprising observation suggests that IgM- and IgG-producing B cells independently entered the intrathecal compartment, and that they further matured and expanded independently of each other in the CSF without class switching. This is in line with the second surprising observation, namely that the AICDA-positive, IgM producing CSFresident B cells that we could identify obviously had not switched to IgG despite expression of AICDA. Together, these observations indicate that the majority of IgM-expressing B cells did not switch to IgG as has been described for certain CD27 + IgM ‘memory B cells’ in peripheral blood (Wu et al., 2010). Indeed, it is well documented that CSF-resident B cells from multiple sclerosis patients are positive for CD27 (Corcione et al., 2004; Cepok et al., 2005). Whether or not these IgM + CD27 + memory B cells depend on T cell help (Weller et al., 2004; Colombo et al., 2013) remains an unresolved question. Our clonal tracking experiments (Fig. 4) indicate that IgG and IgM producing B cells continue affinity maturation, i.e. SHM, after having entered the intrathecal compartment from the periphery. They presumably belong to the CD27 + B cell subset known to be present in blood (Weller et al., 2004; Seifert and Kuppers, 2009;

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Figure 6 Analysis of transcripts of IgM, IgG, and AICDA in single cells from CSF of patients with multiple sclerosis. (A) Agarose gel electrophoresis of PCR products of IgG, IgM and AICDA. We show representative gels of multiplex PCR products from five single cells (sc1 to sc5) isolated from Patient MSM-11. All PCR products were cloned and full-length sequenced. Next to the lanes showing the Ig- and AICDAspecific products, size markers are shown. Sizes are given in the right column (bp). (B) Deduced amino acid sequences of the IgM- and IgGheavy chain PCR products shown in Fig. 5A. Column 1 gives the cell number, column 2 the family of the heavy chain VH, and column 3 the amino acid sequence. We included the first 24 nucleotides of the C-regions to confirm the Ig families. See legend to Fig. 1 for details.

Identification of the target antigens of the intrathecal IgM and IgG responses remains a major challenge for future studies.

Acknowledgements We thank Joachim Malotka for expert technical assistance, Ramit Mehr for providing IgTree, Edgar Meinl and Naoto Kawakami for valuable comments on the manuscript, and the Biobank La Fe, Valencia, Spain, for providing patient samples.

Funding This work was supported through grants from the Deutsche Forschungsgemeinschaft (CRC-TR-128-A5, -128-B8, and the Munich Cluster for Systems Neurology (SyNergy), the European Molecular Biology Organization (ASTF 449-2010), the European Neurological Society, the Instituto de Salud Carlos III (FIS 09/ 00551), the Bundesministerium fu¨r Bildung und Forschung (Kompetenznetz MS, KKNMS), and the University of Ulm (Bausteinprojekt).

Supplementary material Supplementary material is available at Brain online.

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