Fingolimod alters intrathecal B cell maturation in multiple sclerosis patients

Background : B cells are postulated to play multiple roles in the pathogenesis of multiple sclerosis (MS) including secretion. Natalizumab and fingolimod are effective MS therapies that disrupt lymphocyte migration but exert differential effects on B cell maturation and trafficking. Herein, we investigated their effects on peripheral blood and cerebrospinal fluid (CSF) B cell repertoires. Methods : Paired CSF and peripheral blood (PB) lymphocytes were collected from MS patients at baseline and after 6 months of treatment with fingolimod (n = 4) or natalizumab (n = 4). B cell subsets including naïve, CD27+ memory, CD27-IgD- double-negative B cells and plasmablasts were collected by FACS and their respective heavy-chain variable region (VH) repertoires assessed by next generation deep sequencing (Illumina MiSeq). Results : Treatment with fingolimod lead to a distinct contraction of the PB B cell pool whereas natalizumab resulted in an expansion of circulating PB B cells. In contrast, CSF B cell numbers remained stable under treatment with fingolimod but decreased following natalizumab therapy. Clonal overlap between CSF and peripheral blood B cells was reduced following natalizumab treatment (-24% reduction of clonal groups) but remained stable with fingolimod therapy. Lineage analyses of CSF B cell repertoires at baseline and following therapy revealed large, clonally expanded B cell clusters in natalizumab-treated MS patients but no intrathecal clonal expansion following fingolimod therapy. Conclusions : Our findings suggest that natalizumab treatment diminishes the exchange of peripheral and intrathecal B cells but does not impact intrathecal clonal expansion. In contrast, fingolimod treatment fails to alter B cell exchange across the blood-brain-barrier but affects intrathecal clonal expansion. Sphingosine-1 phosphate receptor inhibition may impact MS disease progression by inhibiting intrathecal germinal center activity . blood B cell numbers and CSF B cell clonal expansion without diminishing B cell migratory potential. patient CSF and serum analysis at baseline and 6-months post treatment with either fingolimod or natalizumab (Ig mass sequencing cohort). peripheral blood B cells. This indicates that B cells still enter the CSF under fingolimod treatment but might not undergo further somatic hypermutation and clonal expansion within the CSF compartment. Looking at the composition of B cell subsets in clonal groups between

immunoglobulin G (IgG) bands is a fundamental hallmark of disease and associated with worsening disability [5,16]. Phase 3 clinical trials have demonstrated that B cell-depleting therapies are effective in reducing clinical and MRI activity in relapsing and progressive forms of disease [9,10,24].
Oral fingolimod and natalizumab are MS therapeutics that interfere with lymphocyte migration and exert distinct effects on B cell trafficking [11,27]. Fingolimod is a structural analogue to sphingosine and acts as a superagonist for the sphingosine-1-phosphate receptor 1 (S1P1) on lymphocytes [22] thus inhibiting the egress of lymphocytes from secondary lymphoid tissues. Fingolimod treatment produces a significant peripheral lymphopenia with decreased B cells [23]; the CSF B cell fraction, however, remains elevated [14]. Natalizumab, a monoclonal antibody against α4-integrin, reduces the ability of activated T cells to migrate through the blood-brain barrier, producing significantly reduced B cell numbers in the CSF. Following natalizumab treatment, peripheral blood B cells are increased due to the release of memory B cells that are normally attached to the marginal sinus of the spleen via alpha 4 integrins [14,20,31].
The purpose of this study was to compare and contrast CNS B cell trafficking and distribution in natalizumab-and fingolimod-treated MS patients using massively parallel DNA sequencing of immunoglobulin heavy chain (VH) repertoires. In total, 8 patients receiving either natalizumab or fingolimod were studied at baseline (T0) and following 6 months (T6) of treatment. At each time point, CSF and peripheral blood B cells were sorted into naïve, memory, double-negative and plasmablast populations and VH repertoires assessed by next generation mass sequencing. Comparison of Ig repertoires between the peripheral blood and CSF compartments at baseline and over time indicated that natalizumab treatment diminished but did not completely block the migration of B cells into the CSF. In contrast, fingolimod significantly reduced peripheral blood B cell numbers and CSF B cell clonal expansion without diminishing B cell migratory potential.

Standard protocol approvals, registrations and patients
Patients with relapsing remitting multiple sclerosis were recruited into the study at the University of Colorado, School of Medicine from 2013-2014 following Institutional Review Board-approved informed consent. Study inclusion criteria were the following: 1) diagnosis of clinically definite MS according to International (McDonald 2011) or Poser criteria; 2) age between 18-65 years of age; 3) the ability to give informed consent; and 4) normal vital signs. Exclusion criteria included 1) CNS disease in addition to MS; 2) primary progressive or secondary progressive forms of MS; 3) bacterial or viral infection within the last 30 days; 4) prior immunomodulatory or immunosuppressant therapy with natalizumab, oral fingolimod, azathioprine, methotrexate, cyclophosphamide, mitoxantrone, rituximab, daclizumab, mycophenolate mofetil, laquinimod, oral fumarate, or total body irradiation; and 5) treatment within the last 30 days with corticosteroids, beta-interferon, glatiramer acetate, plasma exchange, or intravenous immunoglobulin. The planned analysis was for 8 patients: 4 natalizumab and 4 oral fingolimod. Nine patients were recruited; a single patient withdrew from participation after initial lumbar puncture. Treatment with oral fingolimod or natalizumab was initiated at the discretion of the patient's treating physician. The study duration was 6 months with peripheral blood and CSF obtained at study entry and exit. Additional fluorescence-activated cell sorting (FACS) analyses were performed on peripheral blood B cells from untreated, natalizumab-treated, and fingolimod-treated MS patients (15 per cohort) at the Technical University of Munich following Institutional Review Board-approved informed consent. Clinical and demographic data for each patient cohort is presented in supplementary materials (Suppl. Table 1).
Separate PCR reactions were performed for each VH-family to avoid cross-priming or primer competition. Each nested Ig constant region primer contained a sequence tag ("barcode") to identify the specific patient B cell population of origin and Ig isotype, a random sequence insert (unique molecular identifier; UMI) to identify PCR over-replication [13], and a unique shared primer sequence

Data analysis
According to their barcodes, Illumina sequencing reads in FASTQ format were pre-sorted into patientspecific B cell populations. Illumina MiSeq high-throughput sequencing reads were quality-controlled, assembled, and filtered using pRESTO [34]. The workflow took advantage of our unique molecular identifiers (UMIs) to eliminate redundant reads from the same PCR priming event.
The nucleotide CDR3 sequence identity. From identified clonal groups within the same patient populations, lineage analysis (rooted B cell lineage trees) was performed using IgTree [2] (kindly provided by Prof. Ramit Mehr, Bar-Ilan University, Tel Aviv) to sort clones based on their mutational distance from germline. Alignments were initiated at nucleotide 75. The germline used as the root of each clonal tree was determined by automatically searching for the best match of the V and J alleles from the IgBlast genes (Blastn v2.2.31), concatenating both alleles into a sequence (G) that was then aligned to each member of the clonal group. Because the D segment is often difficult to determine, we filled in the missing gaps of the CDR3 region in the sequence (G) by Ns in order to avoid hypervariable region bias in IgTree.
Each clonal group tree was then converted to a graphml file using in-house conversion tools and improved via Cytoscape functionalities. The program GNU parallel was used at multiple steps in the assembly of full-length VDJ sequences. Using the output of IgTree, it was also possible to evaluate the relatedness of Ig transcripts over time (T0 vs. T6) and across compartments (peripheral blood vs.

CSF).
Protocol for tree sorting and detailed lineage analyses The IgTree program produces, in addition to numeric images, ".vsdot" files. Those files contain descriptive information for each individual tree. Using in-house python scripts, we extracted the tree structure and composition. All trees were then automatically sorted step by step as described in Suppl. Table 3 and then manually refined. After the refinement, nodes in the lineage tree were labeled based on the compartment (peripheral blood or CSF) and B cell subtype of the most frequent sequence in that clonal group.

Statistics
Wilcoxon signed-rank test (one-sided) for paired samples was used to test for significance between different time points within treatment groups. The Mann-Whitney U test was used to test for significant differences between treatment groups. For comparison of different B cell subsets the nonparametric Kruskal-Wallis test with multiple comparison correction (Dunn's procedure) was applied.

Clinical and laboratory data
Nine patients from the Rocky Mountain Multiple Sclerosis Center at Anschutz Medical Campus were recruited for participation based on inclusion/exclusion criteria and signed informed consent. One patient withdrew after the initial lumbar puncture. MS treatment was pre-determined by the patient's treating physician. Two natalizumab patients and one fingolimod patient were treatment naïve; all initiated therapy within roughly 1 year of their initial presentation. The remaining five patients had received prior MS treatments including beta-interferon, glatiramer acetate, natalizumab, and dimethyl fumarate. Five patients (3 fingolimod and 2 natalizumab) switched therapy due to disease activity.
Two changed treatments after clinical relapses (2 and 6 months, respectively), while the others switched due to new disease activity on MRI. Both relapses occurred while off of therapy. No patients experienced a clinical relapse during the 6-month study period. All standard of care brain MRIs performed 12 months after treatment did not show any new lesion activity (further clinical data Supplemental Table 1). Although 2 patients from the natalizumab treatment group showed a mild CSF pleocytosis at the onset of treatment, there were no significant difference in any baseline CSF parameters between treatment groups (Table 1). There were no significant differences in the changes from baseline in any CSF parameter between treatments (Table 1).
We examined alterations in peripheral blood B cell subsets in an independent cohort (TU-Munich, Suppl. Table 1 Within the CSF, the number of recovered B cells did not change appreciably from baseline following fingolimod therapy (Suppl.  Table 2).
At baseline, the distributions of VH isotypes in peripheral blood B cell populations were 50% IgD and 50% IgM in the naïve B cell population; 50% IgM, 30% IgA and 20% IgG in the memory population; and 26% IgM, 53% IgA and 21% IgG in the DN population (not shown). Plasmablasts were 30% IgM, 40% IgA and 30% IgG. In the CSF, at baseline, there was a significantly higher fraction of IgGexpressing B cells in the memory, DN and plasmablast subsets compared to peripheral blood (not shown). There were no significant changes in the distribution of Ig isotypes in the peripheral blood and CSF subsets following treatment with either medication (Suppl. Figure 1A).
Prior to the onset of treatment, the VH germline distribution of peripheral blood B cell subsets approximated germline prevalence; the VH3 family was most frequently utilized, followed by the VH1 and VH4 family gene segments [3] (Suppl. Figure 1B). The baseline VH germline distribution in the CSF, however, showed a significant increase in VH4 family gene segments consistent with independent findings observed at the single-cell level in MS CSF plasmablasts [25]. Increased VH4 germline segment usage was accompanied by a significant decline in VH1 and VH5 family gene segments. Treatments had no discernable effects on VH family distributions in peripheral blood and CSF (Suppl. Figure 1B). In the CSF, there was a significant increase in Ig class-switched B cells relative to peripheral blood that was accompanied by sharp declines in CSF IgM-and IgD-expressing B cells (Suppl. Figure 1A). Consistent with these results, we observed a significantly lower diversity index for CSF B cells when compared to those in the periphery, indicating, that CSF B cells are predominantly composed of antigen experienced, clonally-expanded populations (Suppl. Figure 1C). Within the CSF compartment, approximately 80% of VH sequences were within clonal populations (  Following treatment, there were considerable changes in the composition of peripheral blood VH repertoires ( Table 2). At baseline, approximately 50% of peripheral blood VH sequences were found within clonal groups with an average clone size of 4 sequences. Following fingolimod treatment, the percentage of peripheral blood VH sequences within clones increased to 74% with an average clone size of 10 sequences; whereas following natalizumab treatment, the percentage of clonal VH sequences in the peripheral blood reduced this to 38% with a stable clone size of 4 sequences. The average clone size in the peripheral blood memory B cell populations increased following fingolimod treatment (T0 = 5, T6 = 14 sequences/clone, p = NS) and decreased (T0 = 4, T6 = 3 sequences/ clone, p = 0.04) following natalizumab therapy (data not shown).

Clonal relationships between peripheral blood (PB) and CSF repertoires at baseline and 6 months following treatment
The prevalence of CSF VH sequences identified in paired peripheral blood repertoires at the onset of therapy was 34.8% in fingolimod-and 30.5% in natalizumab-treated patients (range: 6.5% − 49.8%) ( Table 3). After six months, neither treatment significantly altered this overlap. The percentage of blood VH sequences identified in the CSF was considerably lower, consistent with the increased diversity of the peripheral blood repertoire (Table 3). Comparing between therapies, there was an increase in the fraction of overlapping PB and CSF VH sequences in fingolimod-treated relative to natalizumab-treated patients (Table 3). Table 3 Overlap of VH repertoires between and within the peripheral blood and cerebrospinal fluid in treatment cohorts.  Table 3: Description of sorting guidelines for the development of clonal maturation trees. Supplemental Table 4: Lineage analysis of clonal groups between the CSF and PB. Supplemental Table 5: Lineage analysis of clonal groups between baseline (T0) and after 6 months of treatment (T6) compartments ( Table 3). The percentage of baseline CSF sequences identified following 6 months of treatment was limited following either treatment but was lower in fingolimod-treated subjects (0.3% vs. 6.2%). Even within the highly clonal CSF plasmablast repertoires, the persistence of baseline clones was reduced to 0.1% overlap in the fingolimod-treated patients versus 8.3% overlap in natalizumab-treated patients (data not shown). A similar significant treatment-specific effect was noted in the persistence of PB clones. Again, fingolimod-treated subjects displayed a significant longitudinal reduction in overlapping sequences relative to natalizumab-treated subjects (0.7% vs. 5.5%) ( Table 3).

Lineage analysis of overlapping VH sequences
We aligned clonally related blood and CSF VH sequences to their most homologous germline sequence, developed hierarchical maturation diagrams (clonal groups) using IgTree, and found 659 of 1019 clonal groups suitable for a qualitative analysis of clonal relationships amongst B cell subsets and compartments. The succession of B cell subtypes and their patterns of somatic hypermutations were then used to predict the most likely direction of maturation (in or out of the CSF). Overall, 388 clonal comparisons were established between CSF and PB at each time point (Fig. 2) and 271 clonal groups within the CSF between baseline (T0) and 6 months of treatment (T6) (Fig. 3). We observed directional B cell trafficking with a blood B cell followed by a CSF B cell in 90% of the trees (Fig. 2, clones A-C, E) whereas 10% of comparisons indicated a likely CSF precursor (Fig. 2, clone D). We further quantified the size of clonal groups by counting the number of contributing sequences within each compartment and B cell subset to determine the likely site of clonal expansion at T0 and T6 ( Fig. 4, Suppl. Table 4).
We first separately analyzed bi-compartmental clonal groups (CG) connecting PB and CSF sequences at baseline or after 6 months of treatment ( Fig. 4A-C, Suppl. Table 4). Although fingolimod treatment did not affect the average number of clones shared between CSF and peripheral blood (T0 = 14 and T6 = 15) there was a clear shift towards PB B cells as the major contributor (T0: 60% of sequences; T6: > 95% of sequences), which was accompanied by a parallel loss in the contribution of expanded CSF B cells (T0: n = 106 to T6: n = 18, -85%), particularly CSF plasmablasts, to common lineages ( Fig. 4B). Together the data indicate that PB B cell trafficking into the CNS was not outwardly altered by fingolimod therapy, but rather clonal expansion of CSF B cells diminished. Indeed, there were significantly fewer number of average CSF sequences at T6 following fingolimod treatment (n = 18) when compared to natalizumab (n = 133, p = 0.03); the difference at T0 between groups was not significant (Fig. 4A). Following natalizumab treatment, there was a modest decline in the average number of clones shared between CSF and circulating B cells (T0: n = 46; T6: n = 35, -24%), but with no apparent shift in the compartmental contributions of CSF (T0: n = 183 to T6: n = 133, -27%) or PB sequences (T0: n = 535 to T6: n = 401, -25%). The major site of clonal expansion (PB, CSF or equally in both compartments) and changes following treatment are summarized in Fig. 4C. Following fingolimod treatment, there was an increase in PB expanded clones (from 74% at T0 to 91% at T6), a decreased contribution of CSF expanded clones (14% at T0 to 5% at T6), and an overall reduction in clones equally abundant in both compartments (14% at T0 to 5% at T6). In natalizumab-treated patients, the distribution of major clonal B cell populations was rather static and consisted mostly of PB memory, DN B cells and plasmablasts ( Fig. 4B and 4C). CSF plasmablasts were still the major CSF contributor to clonality (~ 15%).
Further evidence for impaired B cell expansion within the CSF during fingolimod treatment is indicated from the longitudinal analysis of shared CSF and B cell maturation trees (Fig. 4D-F, Suppl. Table 5). Following treatment, none of the CSF-expanded clones at T0 were identified in the CSF at T6, and only a single clone arising from the CSF DN B cell population was identified in the peripheral blood PB DN and memory B cell subsets. Thus treatment with fingolimod appeared to halt expansion of existing clonal B cell populations within the CSF; some of the remaining clones overlapping with clonal populations evident in the peripheral blood at T0 (Fig. 4E, 4F).
In contrast, on average 4 (total = 11) clonal groups connected the CSF compartment between T0 and T6 following natalizumab treatment with more than 50% of shared T0 clonal sequences (T0: 330, T6: 168) still present. Furthermore, an average of 10 clonal groups (total n = 30) were found connecting CSF at T0 and the PB at T6 (Fig. 4D) with only a limited number of average clonal sequences in PB at T6 (n = 18). CSF plasmblasts, memory, and DN B cells, which were the major contributors to clonality at T0 in the natalizumab-treated cohort, changed only marginally at T6. An equal distribution of clonal expansion was observed between the different time points. These results point towards continued B cell maturation within the CSF compartment in natalizumab-treated patients over time.
In addition, we analyzed clonal groups connecting PB at T0 and PB and CSF at T6 (Fig. 4G, H and I, Suppl. Table 5). The average number of clonal groups between both time points was significantly lower following fingolimod treatment when compared to natalizumab (fingolimod n = 7 vs.  4I) in fingolimod-treated patients was found in the PB at 6 months but was equally distributed between the PB and CSF in natalizumab-treated patients (Fig. 4F).

Discussion
B cells are likely to play multiple roles in the MS pathogenesis [9,24]. In this study we investigated the effects of fingolimod and natalizumab on the migration and clonal expansion of B cells before and after 6 months of treatment in a total of eight MS patients by assessing Ig VH repertoires in the peripheral blood and CSF using next generation sequencing. Our findings suggest that natalizumab treatment might diminish but not completely block the migration of B cells into the CSF; whereas fingolimod significantly reduces peripheral blood B cell numbers (Fig. 1) and clonal populations (Table 2) without affecting the exchange of B cells across the blood brain barrier (Fig. 4). In addition, fingolimod appears to reduce B cell maturation and expansion of migratory B cells within the CSF compartment resulting in depletion of pre-existing clonal populations.
While our analysis was limited by patient numbers and the limited number of recoverable CSF B cells in treated patients, our study provides detailed information on the differential treatment effects of two highly-effective MS therapies on B cell repertoires in the PB and CSF. In agreement with prior publications [14,19], CSF B cell numbers recovered from fingolimod patients remained generally stable but declined in natalizumab treated patients. As anticipated, fingolimod reduced the number of circulating B cells in the PB while natalizumab resulted in an increase in PB B cells [14,31,32]. The distinctive contraction and expansion of the circulating PB B cell population by fingolimod and natalizumab may have influenced the ability to detect related sequences in the blood and CSF compartments.
Our results at baseline (T0) are comparable to recent analyses of CSF and peripheral blood VH repertoires in untreated MS patients [26,30,35]. In these studies, clonal relationships were observed between CSF B cells and peripheral (class switched) memory, double negative B cells and plasmablasts [26], and the patterns of mutations in VH sequences in bi-compartmental clones are consistent with migration of B cell populations across the blood-brain-barrier accompanied by expansion within the CNS. In our analysis, variable degrees of overlapping sequences were identified based on the comparator population employed. Before treatment, the overlap between the PB and CSF in our 8 patients was 3.8% using the blood repertoires as a comparator or 34% when using CSF repertoires as the comparator. In previous studies, the average percentage of overlap was lower, with only 6.3% (range 0.6% − 23.5%) of the CSF B cells detected in the peripheral blood repertoire [35].
The distinctions are likely due to the different processing of Ig repertoires, the definition of clones, distinct patient populations, and different depths of mass sequencing. Similar to prior bicompartmental MS B cell repertoire analyses [26,30,35], lineage analyses of clonal groups between the CSF and blood compartment in our patients suggested a bidirectional exchange of B cells across the blood-brain-barrier. In contrast to previous studies [26], we also found a limited overlap of PB naïve B cells to the CSF compartment (< 10%) which could be explained by a greater depth of sequencing in our analyses; memory, DN B cells and plasmablasts from the peripheral blood all contributed approximately 30% to clonal groups emanating from PB.
After 6 months of therapy with either fingolimod or natalizumab, we observed distinct effects on the quantitative overlap of sequences and B cell lineage trees (clonal groups) between the PB and CSF compartments although results have to be interpreted with care due to the limited number of patients. Following fingolimod treatment, the percentage overlap of PB derived sequences to the CSF repertoire increased while the number of linked CSF and PB clonal groups remained stable (Fig. 4A).
In contrast, natalizumab treatment reduced both the percentage overlap of PB derived sequences to the CSF repertoire and the number of linked clonal groups (Fig. 4A). This reduction following natalizumab treatment is consistent with its known effects on restricting activated immune cell trafficking to the CNS. The high frequency of clonal groups shared between PB and CSF under fingolimod treatment suggests that despite the limited number of PB B cells CSF migration remains unaffected. This is consistent with previous observations demonstrating a relatively high fraction of B cells in the CSF of patients receiving fingolimod therapy [14,19]. In the current study, the increased overlap between the peripheral blood and CSF B cells in fingolimod-treated patients, may be magnified by the significant contraction of the circulating PB B cell population.
In addition, our results suggest that fingolimod may inhibit the lifespan of B cells or germinal center activity within the CNS. In our longitudinal analyses of CSF and blood repertoires pre-and posttreatment, we noted a lower amount of overlapping sequences and clonal populations in fingolimodtreated patients when compared to natalizumab-treated or untreated MS patients. Furthermore, detailed lineage analyses revealed that clonal expansion was significantly reduced compared to that observed following natalizumab treatment (Fig. 3). The limited detection of clonal groups connecting the peripheral blood compartment between T0 and T6 in fingolimod-treated patients may have been due to sequestration of PB B cells in peripheral lymphoid tissue. However, no clonal groups persisted amongst pre-and post-treatment CSF B cells under fingolimod treatment at all. In addition, after 6 months of fingolimod treatment, the number of clonal groups connecting the peripheral blood and CSF remained stable but showed a smaller contribution from CSF B cells and a larger contribution from clonally-expanded peripheral blood B cells. This indicates that B cells still enter the CSF under fingolimod treatment but might not undergo further somatic hypermutation and clonal expansion within the CSF compartment. Looking at the composition of B cell subsets in clonal groups between the PB and CSF compartment at T0 and T6, the percentage of CSF plasmablasts was reduced under fingolimod treatment, indicating that the occurrence of newly emerging antibody producing plasmablasts might be inhibited by the drug. Although circulating memory B cells are decreased in the peripheral blood of fingolimod-treated MS patients, memory B cells appear to maintain an intensive exchange across the blood-brain-barrier.
Our results on persisting clonal groups in the CSF of MS patients show some similarities to a recently published paper examining treated and untreated MS patients [6]. In general, the number of persisting clonal populations were lower than in our study (range 1-9) and were preferentially found in patients who experienced relapse during the observational period ( times less than in serum [8]. In contrast, natalizumab is more likely to affect B cell trafficking across the blood-brain-barrier in agreement with recent studies in EAE models [18]. Phosphorylated FTY720 (fingolimod) readily crosses the blood-brain-barrier and is capable of interacting with CNS-resident cell populations [17,28]. Previous work in the mouse experimental autoimmune encephalomyelitis (EAE) model has shown that fingolimod becomes ineffective in mice selectively deficient for sphingosine 1-phosphate 1 receptors on astrocytes [4]. In addition, fingolimod ameliorates chronic progressive experimental autoimmune encephalitis mediated by astrocytes, microglia and pro-inflammatory monocytes [28], and mice treated with fingolimod demonstrate a significant reduction of germinal center activity in peripheral lymphoid tissues [7]. To the best of our knowledge, there are no directly comparable data regarding the effects of fingolimod on CNS germinal center activity in humans.
Our analysis of the overlap between the CSF and peripheral blood B cells in MS patients under fingolimod and natalizumab treatment has several limitations. First, we evaluated only four patients in each treatment group limiting the power of our analyses and leaving some results at a descriptive level. Despite this limitation, we were still able to detect significant differences between treatment groups. Second, we observed a more intense overlap between the PB and CSF compartment at baseline in MS patients receiving natalizumab. While these differences were not significant, physician biases and disease activity may have biased patient selection into the treatment groups thereby influencing the results. Indeed, natalizumab treated patients showed a shorted disease duration and lower age; however, EDSS values were only slightly higher in the natalizumab cohort (Suppl. Table 1).
Nevertheless, we were able to generate representative repertoires in both treatment groups, and our within the naïve B cell pool [33]. We have worked to minimize any such bias by validating each individual Ig lineage tree according to B cell subsets, distance to germline, and time point of analysis.
In summary, our results confirm that natalizumab inhibits but does not completely block the migration of B cells across the blood-brain-barrier. Natalizumab, however, does not impair B cell germinal center activity within the CNS. While the exchange of PB B cells across the blood-brain-barrier does not seem to be inhibited following fingolimod treatment, B cell numbers are significantly reduced in the peripheral blood. More importantly, B cells seem to be inhibited in their ability to undergo further maturation within the CNS. Especially in the context of progressive MS, in which ongoing germinal center activity within the CNS may be compartmentalized [29], CNS penetration of sphingosine-1phosphate (S1P) inhibitors may offer a unique avenue to modulate the intrathecal B cell response.
Although fingolimod treatment did not reach primary end-points in the INFORMS trial in primary progressive MS, MRI parameters of disease activity showed a significant reduction in the number of T2 lesions [21]. Recently, siponimod, a more selective S1P receptor functional antagonist demonstrated benefit in the treatment of SPMS [12] and was recently approved by the FDA. Thus, S1P

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the first author on reasonable request.

Competing interests
MCK: receives financial support (travel compensation, IIT, scientific advisory boards) from Merck,     Lineage analysis of the clonal groups (CG) identified in both peripheral blood (PB) and cerebrospinal fluid (CSF) compartments were analyzed at baseline (T0) and after 6 months