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Fingolimod therapy modulates circulating B cell composition, increases B regulatory subsets and production of IL-10 and TGFβ in patients with Multiple Sclerosis
Journal of Autoimmunity, June 2016, Pages 40 - 51
Fingolimod, an oral therapeutic agent approved for patients with relapsing-remitting Multiple Sclerosis (MS), has been shown to prevent lymphocyte egress from secondary lymphoid tissues; however the specific drug effect on B cells in fingolimod-treated patients remains to be fully elucidated. We present here a comprehensive analysis on the proportions of B cell subsets in the periphery, and the levels of activation, functional surface markers and cytokine profile of B cells in MS patients, following initiation of fingolimod therapy, using flow cytometry and cytokine bead array. Fingolimod therapy increased the ratio of naïve to memory cells, elevated the percentage of plasma cells and highly increased the proportion of transitional B cells as well as additional regulatory subsets, including: IL10+, CD25+ and CD5+ B cells. The percentage of activated CD69+ cells was highly elevated in the remaining circulating B cells, which produced increased levels of IL10, TGFβ, IL6, IL4, LTα, TNFα and IFNγ cytokines, with an overall increased ratio of TGFβ to pro-inflammatory cytokines. Furthermore, fingolimod therapy reduced ICAM-1+ cells, suggesting a possible reduction in antigen-presenting capacity. Phosphorylated-fingolimod was shown in vitro to reduce S1PR1 RNA and protein, to slightly increase viability and to activate anti-apoptotic Bcl2 in transformed B cells of patients with MS. In conclusion, fingolimod therapy modulates significantly the composition of circulating B cells, promoting regulatory subsets and an anti-inflammatory cytokine repertoire.
- Fingolimod modulates B cell composition and functional markers in patients with MS.
- Fingolimod reduces memory B cells, while increases transitional and plasma cells.
- Fingolimod elevates B regulatory subsets, such as IL10, CD25 and CD5 B cells.
- Remaining circulating B cells are activated, showing increased cytokine production.
- Fingolimod promotes elevated ratios of TGFβ to pro-inflammatory cytokines.
Keywords: Autoimmunity, B cell subsets, Cytokine, Fingolimod, Multiple sclerosis, Regulatory cells.
Multiple Sclerosis (MS) is considered a predominantly T-cell mediated autoimmune disease; however additional cell types, including B cells, contribute to disease pathology . Emerging evidence of the role of B cells in MS pathology includes the presence of oligoclonal immunoglobulins in the CSF and of memory and plasma B cells within MS lesions from onset of clinical symptoms  and , the presence of a demyelinating lesion pattern characterized by specific antibodies and complement components in subgroups of MS patients , the identification of B-cell follicle-like structures in the MS brain meninges , and most convincingly the highly beneficial results from clinical trials of selective B-cell depleting therapies such as Rituximab and Ocrelizumab , , and .
Fingolimod (FTY720, Gilenya®) is an approved oral disease modifying treatment (DMT) for relapsing-remitting patients with MS (PwMS). FTY720 (FTY) is phosphorylated within the body by sphingosine kinases to the biologically active phosphorylated-fingolimod (PFTY), a structural analog of Sphingosine 1-phosphate (S1P). S1P is involved in several cellular processes and mediates lymphocyte trafficking from secondary lymphoid organs (SLOs) to the circulation, along a S1P gradient. The beneficial mechanism of fingolimod is mainly attributed to its effect as a functional antagonist for one of the five S1P receptors: S1PR1  and . Binding of fingolimod to S1PR1 causes internalization and degradation of the receptor, thereby preventing cells from responding to the S1P gradient, retaining lymphocytes within SLOs . The consequent reduction of circulating lymphocytes is believed to reduce the infiltration of autoreactive and pathogenic lymphocytes into the CNS.
Data on fingolimod therapy affecting B cells is only beginning to emerge , , , and . The goal of this study was to provide a comprehensive analysis on the spectrum of B cell subsets, the levels of activation and functional markers, and the cytokine profile of B cells in a 3 month (m) follow-up study in patients initiating fingolimod therapy. Furthermore, the functional effect of FTY and PFTY on selected markers was assessed also in vitro in B cells from treatment-naïve PwMS.
2. Materials and methods
2.1. Study participants
Relapsing remitting (RR) MS patients (>18 years) fulfilling the revised McDonald criteria  were recruited at the MS center at Carmel Medical Center, Israel, after providing written informed consent and with the approval of the local Helsinki committee. For the ex vivo study, 36 RRMS patients were recruited and blood samples obtained before and 3 m after initiating fingolimod therapy. Patients were free of prior DMT or steroid treatment for at least 1 m, in remission, with Expanded Disability Status Scale (EDSS) ≤6. For the in vitro part, blood samples were obtained from 14 treatment-naïve RRMS patients, without DMT for at least 6 m, or steroid treatment for 1 month, EDSS ≤6, in remission. In some in vitro experiments requiring large cell-numbers, lymphoblastoid cell-lines (LCLs) prepared from 15 treatment-naïve RRMS patients were used as a surrogate model for B cells.
3. Isolation of B cells and culture
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Novamed) and B cells isolated by negative selection (EasySep kit (Stemcell)) or the Human B cell isolation kit II (Miltenyi Biotec), with a purity>90%. Cells were immuno-stained immediately or cultured in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS), penicillin-streptomycin-nystatin (100 U/ml) and l-glutamine (2 mM) (Biological Industries) in a 37 °C humidified 5% CO2 incubator. For in vitro experiments, cells were cultured with or without 10 nM fingolimod (FTY or PFTY, Novartis), a similar dose to the physiological levels reached in the serum of treated patients (10 nM = 3.4 ng/ml) . 10 μg/ml αIgM (SouthernBiotech) and 1 μg/ml αCD40 (BioLegend) were added for B cells stimulation where appropriate. EBV-transformed LCLs were cultured in log phase and used within 1 m of thawing.
4. Flow cytometry
PBMCs were stained immediately with PE-anti-CD14, PerCp-anti-CD3, APC/Cy7-anti-CD4 and FITC-anti-CD19 (Biolegend) for immune subsets. For B cell subsets, cells were stained with FITC-anti-CD19, BV421-anti-CD27, Pe/Cy7-anti-IgD, PE-anti-CD24, APC-anti-CD138 and PerCP-anti-CD38 (Biolegend) for naïve (CD19+CD27-IgD+), memory (CD19+CD27+IgD-), transitional (CD19+CD27−CD38hiCD24hi), plasmablasts (CD19+CD27+CD38+), plasma cells (CD19+CD138+). For B regulatory subsets, cells were stained with APC-anti-IL10, Pe/Cy7-anti-CD5, PerCp/Cy5.5-anti-CD25, and Pacific blue-anti-CD86 (Biolegend). For evaluation of antigen presenting molecules, cells were stained with Pacific blue-anti-HLA-DR, APC-anti-CD40, PE-anti-ICAM-1 and co-stimulatory molecules with Pacific blue-anti-CD86 and PE-anti-CD80 (Biolegend). APC/Cy7-anti-BAFF-R and PerCP/Cy5.5-anti-CD69 (Biolegend) were used for assessment of B cell activation. In the in vitro study, cells were stained after 24 h culture with fingolimod. For intracellular staining of cytokines, αIgM/αCD40-stimulated B were cultured for 40 h, re-stimulated 4 h before end of experiment by 10 ng/ml Phorbol 12-Myristate 13- Acetate (PMA) + 1 μg/ml Ionomycin (Sigma Aldrich) in the presence of Golgistop (BD Bioscience), then stained with FITC-anti-CD19, BV510-anti-CD27, Pe/Cy7-anti-IL4, PE-anti-Lymphotoxinα (LTα), PerCP/Cy5.5-anti-IFNγ, BV421-anti-TGFβ and BV650-anti-TNFα (BD Bioscience) using a Fix & Perm kit (Invitrogen). In the in vitro study, 10 nM fingolimod was added to culture every 24 h to assure continued effect. Unstained cells and appropriate isotype controls were used as negative control for staining, and BD CompBeads (BD Bioscience) used for compensation. Cells were read on an ADP CyAn flow cytometer (Beckman Coulter) or on LSRFortessa (BD bioscience) and results analyzed using FlowJoX. For the ex vivo experiments, Cytometer Setup & Tracking beads (CS&T beads) (BD Bioscience) were used at baseline and at 3 m in order to keep the cytometer performance consistent.
The effect of fingolimod on B cell viability was assessed using a XTT-based colorimetric kit (Biological Industries). LCLs were cultured with 0, 1, 10, 100 or 1000 nM FTY or PFTY for 24 h and viability quantified by spectroscopy.
4.2. Cytokine secretion
Cytokine levels were assessed using the Human Th1/Th2/Th17 Cytometric Bead Array (CBA) kit (BD bioscience) according to the manufacturer's protocol on supernatant from B cells cultured for 24 h with αIgM/αCD40. Analysis was performed on an LSRFortessa (BD bioscience).
5. Quantitative real-time PCR (RTPCR
mRNA was extracted from 2 × 106 LCLs cultured 24 h with or without 10 nM of FTY or PFTY using the high pure RNA isolation kit (Roche) and cDNA synthesized using M-MLV reverse transcriptase (Promega) with random hexamer primers (Biological Industries). RTPCR was performed using SYBR green (Roche) on a Prism® 7300 Sequence Detection System (Applied Biosystems). UBE2D2 was used as reference gene. Primers: S1PR1 forward: TCTGCGGGAAGGGAGTATGT. S1PR1 Reverse: CGATGGCGAGGAGACTGAA (IDT). Relative quantification of mRNA expression was calculated using the comparative CT method .
6. Western Blot (WB) analysis
5 × 106 LCLs were cultured with or without 10 nM FTY or PFTY for 24 h (S1PR1) or for 30, 60 and 120 min (Bcl2), then whole cell lysate prepared and WB performed according to standard methods, using anti-S1PR1, anti-Bcl2 or anti-pBcl2 (Santa Cruz). Samples were normalized according to β-Actin and quantification of relative expression levels performed using Totallab TL100 V2006c software.
6.1. Statistical analysis
Statistical analysis was performed using SPSSv22. Paired T-test was used to compare cells with or without fingolimod treatment (in vitro) and paired T-test or Wilcoxon signed-rank test used to compare data before and after 3 m therapy in the ex vivo experiments, according to the normality of the data as assessed by Kolmogorov-Smirnov/Shapiro-Wilk test. A p-value<0.05 was considered significant.
In total, 36 PwMS were recruited before initiation of fingolimod treatment, and PBMCs and B cells collected at baseline and following 3 m therapy, while EDSS and relapses were reported at 0, 3 and 12 m therapy. Additionally, for in vitro assays, B cells were obtained from 14 PwMS, and LCLs from 15 treatment-naïve PwMS were used as a B cell model. The clinical data on all patients is presented in Table 1. So far, 27 patients have finished 12 m therapy, and no relapses have been reported.
|Patient ID||Age (years)||Gender||Disease duration (years)||EDSS baseline||EDSS 1 year treatment||DMT prior to fingolimod|
|Cell type||Number of patients (Average)||Age (years) (Average)||Gender||Disease duration (years) (Average)||EDSS (Average)|
|Primary B cells||14||39||10F/4M||1.9||2.5|
7.1. Fingolimod treatment alters the proportions of T cells, B cells and monocytes within PBMCs
In order to determine the effect of fingolimod treatment on PBMCs composition, PBMCs were compared at baseline and following 3 m therapy in 10 PwMS, results are shown in Fig. 1. The percentages of CD3+CD4+ T cells (p < 0.0001) and CD19+ B cells (p < 0.0001) were significantly reduced after 3 m, while the proportion of CD14+ monocytes increased (p < 0.0001) and the proportion of CD3+CD4− T cells (mainly CD8+ T cells) remained unchanged. The absolute cell counts of CD3+CD4+ T cells, B cells and to a lesser extent, CD3+CD4− T cells were also significantly decreased, while no change was seen on the absolute count of monocytes (data not shown).
7.2. Fingolimod affects B cell viability and S1PR1 expression
Since fingolimod has been shown to affect apoptosis , we assessed if fingolimod affects B cell viability by XTT, using LCLs cultured 24 h with or without increasing dose of FTY or PFTY. 1–1000 nM FTY slightly but significantly reduced cell viability (88.7 ± 3% by 10 nM, p = 0.0006), while in contrast, PFTY slightly increased viability (112 ± 4.5% by 10 nM, p = 0.04); no dose response was observed (Fig. 2A). We next assessed the effect of fingolimod on activation of the anti-apoptotic factor Bcl2, which has previously been associated with FTY-induced apoptosis in other cell types . LCLs were cultured in the absence or presence of 10 nM FTY or PFTY for 0–120min and the levels of the active phosphorylated Bcl2 (pBcl2) and the non-active Bcl2 assessed by WB. PFTY significantly increased pBcl2 within 30 min (137.5 ± 15%, p = 0.034), while FTY had no significant effect (Fig. 2B). This result, together with the small increased viability shown in Fig. 2A, suggests that 10 nM PFTY has a minor anti-apoptotic effect on B cells. The small magnitude of this effect could be a result of using transformed B cells (LCLs), which are highly proliferative.
Fingolimod has been shown to cause internalization of cell-membrane-expressed S1PR1 in T cells . Using LCLs cultured for 24 h with or without 10 nM FTY or PFTY, we found that PFTY significantly reduced S1PR1 expression both on the RNA level (0.77 ± 0.06, p = 0.002) and the protein level (76 ± 8%, p = 0.023), while FTY had no effect (Fig. 2C).
7.3. Fingolimod therapy reduces the percentage of memory B cells and increases naïve and transitional B cells and plasma cells
The effect of 3 m fingolimod therapy on the frequencies of memory B cells (CD27+IgD−), naïve B cells (CD27−IgD+), double negative memory B cells (CD27−IgD-), non-switched memory cells (CD27+IgD+), plasma cells (CD138+), plasmablasts (CD27+CD38+) and transitional B cells (CD27−CD38hiCD24hi), within total CD19+ B cells was assessed in 10 PwMS. The gating strategy is presented in Fig. 3A and results are presented in Fig. 3B. Fingolimod significantly decreased the proportion of memory B cells (p = 0.011) and respectively increased naïve cells (p = 0.011). The proportion of transitional B cells was highly increased from 3.7 to 11.6% (p = 0.005), while no significant change was observed on plasmablasts or on double negative memory B cells and non-switched memory cells (data not shown). In contrast, the percentage of CD138+ plasma cells was elevated (p = 0.03). The absolute cell count of memory cells, and to a lesser extent naïve cells were also significantly reduced, while the absolute number of plasma cells was unchanged (data not shown). Furthermore, the expression (median fluorescence intensity (MFI)) of CD38 (p = 0.008) and CD24 (p = 0.008) was significantly elevated after 3 m therapy (Fig. 3C). A significant reduction in the proportion of memory B cells was also detectable in vitro, in αCD40/αIgM-stimulated B cells treated with either 10 nM FTY or PFTY: FTY- 95.2% ± 1.3, p = 0.005; PFTY -95.3% ± 1.85, p = 0.03 (Fig. 3D).
7.4. Fingolimod alters the expression of markers of antigen presentation and activation in B cells
In order to elucidate whether fingolimod therapy may also affect B cells functionality, we analyzed the levels of several molecules involved in B cell function, including markers associated with antigen presentation, such as CD40, HLA-DR, ICAM-1, CD86 and CD80  and , and B cell activation such as BAFF-R and CD69. Fig. 4 summarizes the results. Although HLA-DR, ICAM-1 and CD40 are expressed in nearly all B cells, 3 m fingolimod therapy caused a slight but significant decrease in the percentage of CD40+HLA-DR+ICAM-1+ B cells (p = 0.036) (Fig. 4AI) and also on their absolute cell count (data not shown). In line with this, 24 h therapy with PFTY in vitro significantly decreased the percentage of ICAM-1+ cells (92% ± 2.7, p = 0.024) (Fig. 4AII). These results suggest that fingolimod could reduce antigen presenting capacity. A minor reduction in the percentage of BAFF-R+ cells (p = 0.035) (Fig. 4B) was seen after 3 m therapy, while the percentage of CD69+ cells (p = 0.007) (Fig. 4C) and the expression (MFI), but not percentage, of co-stimulatory molecule CD86 (trend, p = 0.05) (Fig. 4D), was elevated, indicating an increase in activated cells. The absolute cell count of BAFF-R+ cells was also significantly reduced, while the absolute number of CD69+ cells was unchanged (data not shown). No effect was found on CD80 (data not shown).
7.5. Fingolimod increases regulatory B cell subsets
Next we analyzed the effect of fingolimod therapy on markers associated with regulatory properties. Several subsets both within naïve and memory cells have been described as B regulatory cells (Bregs), and characterized by markers such as IL10, CD27, CD5, CD25, CD86, CD24 and CD38 , , , , , , , , and . Thus, we compared the level of these markers at baseline and after 3 m therapy (Fig. 5). The proportion of IL10+ cells was increased after 3 m therapy in total B cells (p = 0.01), memory (CD27+) B cells (trend p = 0.05) and naïve B cells (CD27−) (trend p = 0.05). Furthermore, the expression of IL10 (MFI) was increased by 74% in naive cells (p = 0.038) (Fig. 5AI) and by 82% in CD27−CD38+CD24+ B cells (p = 0.038) (Fig. 5AII). In addition, fingolimod therapy increased the percentages of CD25+CD5+ (p = 0.008), CD25+CD86+ (p = 0.04) and CD5+ (p = 0.003) B cells, all phenotypes that have been associated with regulatory B cells (Fig. 5AIII). The absolute cell count of the IL10+ subsets and CD25+CD86+ remained unchanged, while the absolute number of CD25+CD5+ and CD5+ cells were reduced, but by a lower rate than total B cells (data not shown). In line with these results, 24 h in vitro treatment with PFTY increased the percentage of IL10+ cells (110.5 ± 4%, p = 0.03), while FTY, but not PFTY, increased the percentage of CD25+CD5+ (111.8 ± 4.76%, p = 0.04) and CD25+CD86+ (107.64 ± 3.2%, p = 0.047) cells, using stimulated primary B cells (Fig. 5B). All in all, these results indicate that fingolimod therapy increases regulatory B cell subsets.
7.6. Fingolimod alters the cytokine profile in B cells
B cells can produce both pro- or anti-inflammatory cytokines. We assessed the effect of 3 m fingolimod therapy on the cytokine profile in supernatant collected after 24 h from stimulated B cells from 11 fingolimod-treated patients. The levels of 6 cytokines: TNFα, IL10, IL6, IL-2, IFNγ and IL4 were assessed by CBA (Fig. 6A). While IFNγ, IL2 and IL4 were below detection level in most samples, fingolimod therapy significantly increased the level of IL6 (p = 0.0036) and IL10 (p = 0.02), while the increase in TNFα did not reach significance. Since this assay does not take into account the fingolimod-induced alterations in B cell subsets, we also assessed the cytokine levels by intracellular immunostaining and flow cytometry. Cells from 9 patients collected before and after fingolimod therapy were cultured 40 h with αIgM/αCD40 stimulation, then stained for CD19, CD27, TNFα, IFNγ, LTα, IL4 and TGFβ and analyzed by flow cytometry. Cytokine levels in total, naïve and memory B cells are shown in Fig. 6B. Fingolimod therapy significantly increased the percentages of IFNγ+ cells, LTα+ cells and IL4+ cells in total B cells (p = 0.008, p = 0.04 and p = 0.008, respectively) and in memory B cells (p = 0.008 for each). Furthermore, the percentage of TGFβ+ cells was significantly increased in total, memory and naïve B cells (p = 0.008, p = 0.008 and p = 0.02, respectively), while a significant elevation in TNFα+ cells was seen only within memory B cells (p = 0.008). Finally, the frequency of pro-inflammatory TNFα+IFNγ+ cells and anti-inflammatory IL10+TGFβ+ cells were elevated after fingolimod therapy in total B cells (p = 0.008 and p = 0.011, respectively) and in memory B cells (p = 0.008 and p = 0.011, respectively), while no difference was found in naïve B cells (data not shown). The absolute cell counts/ml blood of IFNγ+ and IL4+ B cells were not significantly changed, whereas the number of LTα+ and TNFα+ and TGFβ+ B cells was reduced, but by a lower rate than total B cells (data not shown). Calculating the ratio between anti-inflammatory to pro-inflammatory cytokines (Fig. 7), we found that fingolimod therapy increased the ratio of TGFβ/Ltα in total B cells (p = 0.03), of TGFβ/TNFα in naive B cells (trend, p = 0.05), as well as the ratio of IL4/Ltα in total B cells (p = 0.025) (Fig. 7) and in naive B cells (p = 0.03, data not shown). These results indicate that although both pro- and anti-inflammatory cytokines were elevated after 3 m therapy, the overall cytokine profile may become more anti-inflammatory.
It is becoming evident that B cells serve a role in MS pathogenesis. This role may include production of autoreactive antibodies, abnormal antigen presenting capacity, altered cytokine response and possibly impaired number or function of Bregs, all leading to increased activation of autoreactive CD4 T cells , , and . In this study we aimed to further elucidate if and how fingolimod therapy affects the composition and functionality of B cell subsets.
In line with recent publications  and , we found a significant reduction in the numbers and proportions of CD4+ T and B cells within the periphery, while CD3+CD4− T cells (mainly CD8 T cells) were less affected, and the proportion of monocytes was accordingly increased after 3 m therapy. The differential reduction of immune cells in the periphery has been attributed to the cellular level of CCR7, a lymph node homing-chemokine receptor, reducing mainly naïve and central memory CD4 T cells, B cells and CD56 bright natural killer cells, but not CCR7−CD4+ effector memory T cells, less affecting CD8 T cells, skewing the CD4 to CD8 ratio , , and . The reduction in the percentage of lymphocytes in the periphery was previously reported to be a result of their redistribution rather than depletion . Our result confirms drug adherence in our recruited patients and is in line with the suggested mechanism of action of fingolimod of preventing lymphocytes egress from SLOs, via modulation of the S1PR1 receptor. Indeed, the in vitro study demonstrated that PFTY treatment reduces the expression of S1PR1 on B cells, as has previously been shown in other cell types , , , , , and . This downregulation was detected on the protein level, possibly due to receptor internalization and degradation , but also on the mRNA level, as has been described in T cells . The lack of effect of the non-phosphorylated compound FTY could be due to a too short drug exposure in vitro, not enough for phosphorylation and activation of the compound. In contrast, we found that in vitro treatment with FTY or PFTY had opposite effects on B cell viability, as has previously been shown in fibroblasts, microglia or PBMCs , , and . In accordance with this, we showed that PFTY causes phosphorylation and thus activation of the anti-apoptotic Bcl2 in B cells, as has also been shown in fibroblasts . In fact, the therapeutic application of the anti-apoptotic effect of FTY has been studied in several cancer cells , , and ; however these studies were all obtained at μM doses, thus the relatively minor effect in B cells of nM dose, equal to the physiological serum level in fingolimod-treated patients , might not have biological relevance. FTY is phosphorylated within the body by sphingosine kinases to PFTY, and 12–14 h after administration the concentration of both compounds reaches equilibrium , , and . FTY and PFTY have been reported to cause opposite effects on apoptosis and calcium flux  and , and FTY has been shown in some cases to act independently of phosphorylation or S1PR, mainly as a protein phosphatase 2A activating drug , , , , and . Thus, the mechanism of action of fingolimod seems to include both antagonistic effects on S1PR as well as agonistic activations of S1PR-independent pathways.
Since fingolimod therapy was shown to differentially affect T cell subsets, we analyzed the proportions of B cell subsets after 3 m therapy. Fingolimod reduced the proportion of memory cells, increased the proportions of naïve and especially transitional cells, while no significant change occurred in the proportion of plasmablasts, but plasma cells were elevated. The effect on memory and transitional cells is in line with recent reports after 2w  or 12 m therapy , or reports comparing fingolimod-treated patients to other PwMS  and . Since memory cells express less CCR7 than naïve cells  and , CCR7 expression cannot explain the detected skewed ratio of naïve to memory cells after therapy. However, a lower expression of CCR7 in CD27+CD38+ plasmablasts than in memory cells  may explain why no significant change occurred in the proportion of plasmablasts, in line with a previous report after 2w therapy . Additionally, plasmablasts have lower expression of S1PR1 than naive or memory cells , which could also explain why their proportion was unchanged. A similar increase in the proportions of plasma cells was also seen after 2w therapy , suggesting that this subset is less affected by fingolimod, although plasma cells express high levels of S1PR1 . Whether this is due to lower expression of CCR7 is unknown.
In addition, we found that fingolimod increases the expression of CD38 and CD24. This combination of markers, when co-expressed at high levels in naïve cells is characteristic of transitional cells, a population associated with regulatory capacities, such as high production of the anti-inflammatory cytokine IL10, induction of T regulatory cells and inhibition of Th1, Th17 and CD8 T cells , , and . Thus, the increased proportion of transitional cells, as well as elevated expression of CD38 and CD24 following fingolimod therapy, may suggest that B cells remaining in the periphery include increased percentage of regulatory cells. Indeed, we found a significant increase in IL10+ B cells following therapy, and elevated IL10 expression in both naïve and transitional cells. IL10 downregulates the immune response by reducing production of pro-inflammatory cytokines, expression of co-stimulatory molecules and antigen presentation . Increased IL10+ B cells and transitional cells have previously been reported in studies comparing fingolimod-treated patients to other PwMS  and , but our study is the first to report of increased IL10+ B cell subsets in a follow-up study. Bregs have lately been suggested to play a role in autoimmunity , , , and . Bregs are not yet fully characterized and have mostly been associated with high production of IL10, but appear to include different B cell subsets, both within naïve and memory cells. These subsets have been characterized by expression of different markers including CD25, CD86, CD5 or CD24 and CD38 , , , , , , , , and . We demonstrate here for the first time that fingolimod therapy increases the proportions of several of these regulatory subsets including IL10+, transitional B cells, CD25+ CD86+, CD25+CD5+ and CD5+ B cell populations. The elevation in IL10+ cells is most likely due to the elevated proportion of IL10-producing transitional cells, and of plasmablasts and plasma cells, the main B cell producers of both IL10 and IL35 regulatory cytokines . Our results indicate that fingolimod not only reduces the number of autoreactive lymphocytes in the periphery, but also alters the composition of peripheral B cell subsets, mainly increasing proportions of regulatory B cells, crucial for controlling the immune response. A similar increase has been reported in the frequency of Tregs in Fingolimod-treated patients , , , and . The increased proportions of circulating B and T regulatory cells may enhance their capacity to downregulate inflammation, by elevating their ratio compared to their target cells, and may be an important part of the beneficial mode of action of fingolimod in MS.
Our observation that FTY, but not PFTY, in vitro increased the proportions of CD25+ regulatory subsets may be another example of a S1PR1-independent action of FTY, as previously described.
We found that the remaining circulating B cells after 3 m fingolimod therapy produce more both anti-inflammatory and pro-inflammatory cytokines, despite the relative reduction in memory cells, which in general produce more cytokines than naïve cells  and . As mentioned above, this may be due to the unchanged and elevated proportion of plasmablasts and plasma cells respectively, and indicates that the remaining B cells are in a more activated state, in line with the obtained significant elevation in CD69+ cells. While the elevation in IL10 has previously been reported  and , this is the first study to show an elevation in IL6, IFNγ, IL4, LTα, and TGFβ. Our follow up study did not confirm a previous report of a decrease in TNFα production by B cells in fingolimod-treated patients compared to other PwMS . Bregs are thought to immunosuppress pathogenic T cells through the production of IL-10, IL-35, and TGFβ  and . The ratios of TGFβ to either LTα or TNFα and the ratio of IL4 to LTα were elevated after 3 m therapy, suggesting that the overall cytokine profile becomes more anti-inflammatory. TGFβ can trigger a vast array of regulatory responses such as inhibition of antigen presentation and induction of Tregs  and . The biological consequence of an increase in both pro- and anti-inflammatory cytokines in fingolimod-treated patients is intriguing; however stimulation of TGFβ together with pro-inflammatory IL6 has been shown in myelin-reactive T cells, to abrogate their pathogenic function and increase IL10 production, despite also increasing IL17 . Since B Cells of PwMS have been shown to exhibit aberrant pro-inflammatory cytokine responses , the described change in cytokine profile by fingolimod may be an important part of the beneficial effect of the drug.
Finally, we found the drug to reduce the percentage of ICAM-1+ cells, suggesting that fingolimod might reduce antigen-presenting capacity . We did not detect any effect on other antigen presenting markers such as HLA-DR, or co-stimulatory molecules CD80 or CD40; however CD86 expression was elevated after 3 m therapy (trend). In a 12 m fingolimod follow up study, HLA-DR expression was reduced within 1 m, but this reduction was abolished after 1 y . Furthermore, CD80 and CD86 expression was elevated after 3 m and 6 m respectively, in line with our result on CD86. Further functional studies are needed in order to determine, if indeed fingolimod alters antigen presentation. Fingolimod therapy also caused a minor reduction in the percentage of BAFF-R+ B cells, perhaps affecting B cell survival.
In conclusion, we have shown that fingolimod therapy modulates the composition of B cell subsets, by increasing the ratio of naïve to memory cells and highly increasing the proportion of transitional B cells and several other regulatory subsets, including IL10+ cells. The remaining B cell subsets were relatively activated and secreted elevated levels of cytokines. Fingolimod modulates the cytokines profile of B cells, increasing the ratio of TGFβ and IL4 to pro-inflammatory cytokines. The elevated proportion of regulatory B cell subsets, transitional cells and plasma cells in fingolimod-treated patients may have strong influence on the cytokine milieu and disease progression. Indeed, in recent studies B cell-depleting therapy such and Rituximab or Ocrelizumab, which do not affect plasma cells, were shown to be highly effective in MS , , and  while in contrast Atacicept, blocking plasma cells and late stages of B-cell development, aggravated the disease and trial was discontinued . Thus, our results suggest that fingolimod could be beneficial, beyond MS, also in other immune-mediated diseases in which B cells play a key role. The relatively activated B cells remaining in the periphery may, together with effector memory T cells, provide important immune surveillance and protection in fingolimod-treated patients.
9. Disclosure statement
The authors declare no competing interests.
This study has received financial support from Novartis. We thank Sara Dishon, M.PA., Carmel Medical Center, Haifa, Israel for providing patient care and assistance in clinical data management and Anat Wolkowich, M.Sc, for assistance with coordination of this study.
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a Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
b Neuroimmunology Unit & Multiple Sclerosis Center, Carmel Medical Center, Haifa, Israel
∗ Corresponding author. Multiple Sclerosis Center, Department of Neurology, Carmel Medical Center, Haifa, 34362, Israel.
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