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 , 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 . 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 , 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 post-treatment, we noted a lower amount of overlapping sequences and clonal populations in fingolimod-treated 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 . 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 (9–22 months). Amongst those patients receiving natalizumab or fingolimod (n = 2 for each therapy), persisting clonal populations were noted in only one individual for each treatment. Both of these patients, however, showed clinical and MRI activity over an observation period of more than 12 months, making it difficult to compare with our patients who were clinically and radiologically stable over 6 months. Additional variability likely results from the limited number of patients in both studies and limited number of CSF B cells to be sampled in natalizumab- and fingolimod-treated patients.
The combined effects of fingolimod on the PB and CSF B cell repertoires suggests that intrathecal fingolimod may reduce CNS germinal center activity. Although a role of alpha4 integrins in peripheral lymphoid tissue compartmentalization has been established in animal models , a similar effect of natalizumab on germinal center reactions in the CSF is unlikely. The transit of natalizumab across the blood-brain-barrier as an antibody is limited and CSF concentration levels of natalizumab are 350 times less than in serum . In contrast, natalizumab is more likely to affect B cell trafficking across the blood-brain-barrier in agreement with recent studies in EAE models . 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 . In addition, fingolimod ameliorates chronic progressive experimental autoimmune encephalitis mediated by astrocytes, microglia and pro-inflammatory monocytes , and mice treated with fingolimod demonstrate a significant reduction of germinal center activity in peripheral lymphoid tissues . 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 subsequent analyses, normalized by the total number of obtained sequences, were consistent when comparing the total number of overlapping sequences with the detailed analyses of clonal groups. Third, while lineage analysis provides a unique tool to study B cell trafficking in MS, mass sequencing of B cell Ig repertoires only allows a snapshot of the B cell repertoire at a certain time and does not fully represent the dynamic relationship amongst transiting B cells. This may be further skewed by the process of repertoire reconstruction. Although we optimized our protocol to obtain a maximum recovery of Ig sequences from B cells, efficient amplification of VH sequences from memory and naïve B cells is challenging. In addition, despite safeguards such as high fidelity PCR and UMIs, sequencing errors and over-amplification of transcripts may result in artificial or over-represented B cell clusters or clones. In fact, we observed roughly 50% of naïve B cells in clones containing a small number of mutations from germline (3.5 sequences / clone). This could represent sequencing error or activation within the naïve B cell pool . 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.