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Proportions of circulating transitional B cells associate with MRI activity in interferon beta-treated multiple sclerosis patients

Open AccessPublished:July 14, 2021DOI:https://doi.org/10.1016/j.jneuroim.2021.577664

      Highlights

      • A low proportion of transitional B cells is associated with a high risk of MRI activity in interferon beta-treated MS.
      • Vitamin D3 supplementation does not affect circulating B cell phenotypes.
      • Reduced anti-EBNA-1 IgG levels after 48 weeks associate with a lower risk of MRI lesions.
      • EBV-related antibody levels do not correlate with circulating B cell phenotypes.
      • Multiple prognostic parameters contribute to the risk of new MRI-lesions in a prognostic model.

      Abstract

      B-cells contribute to MS pathogenesis. The association of circulating B-cell phenotypes with combined unique active lesions (CUA) on MRI at 48 weeks follow-up was investigated in 50 interferon beta-treated MS patients. Transitional B-cell proportions were lower in participants with CUA at week 0 and 48 [p = 0.004, p = 0.002]. A decrease in circulating anti-EBNA-1 IgG levels between week 0 and 48 associated with absence of CUA [p = 0.047], but not with B-cell profiles. In a multi-factor model for CUA-risk, transitional B-cell proportions contributed independent from NK/T-cell ratio, change in anti-EBNA-1 IgG, and vitamin D supplementation. Transitional B-cells may predict treatment response in MS.

      Graphical abstract

      Keywords

      1. Introduction

      Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), of which the pathogenesis is driven by a complex interplay of environmental and genetic factors (
      • Dobson R.
      • Giovannoni G.
      Multiple sclerosis - a review.
      ). Although the exact underlying disease mechanisms of MS are not yet fully elucidated, several lymphocyte populations have been associated with its disease process. These include T helper 1 cells (CD4+IFN-γ+ T cell, Th1 cell) (
      • Gutcher I.
      • Becher B.
      APC-derived cytokines and T cell polarization in autoimmune inflammation.
      ), T helper 17 cells (IL-17A+CD4+ T cell, Th17 cell) (
      • Durelli L.
      • et al.
      T-helper 17 cells expand in multiple sclerosis and are inhibited by interferon-beta.
      ;
      • van Langelaar J.
      • et al.
      T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.
      ), natural killer (NK) cells (
      • McKinney E.F.
      • et al.
      A CD8(+) NK cell transcriptomic signature associated with clinical outcome in relapsing remitting multiple sclerosis.
      ;
      • Mimpen M.
      • et al.
      Prognostic value of natural killer cell/T cell ratios for disease activity in multiple sclerosis.
      ;
      • Mimpen M.
      • et al.
      Natural killer cells in multiple sclerosis: a review.
      ), and B cells (
      • Arneth B.M.
      Impact of B cells to the pathophysiology of multiple sclerosis.
      ;
      • Wanleenuwat P.
      • Iwanowski P.
      Role of B cells and antibodies in multiple sclerosis.
      ). Several environmental factors associated with MS onset are believed to modulate the functional or phenotypic characteristics of these lymphocytes, including vitamin D levels (
      • Pierrot-Deseilligny C.
      • Souberbielle J.C.
      Vitamin D and multiple sclerosis: an update.
      ;
      • Smolders J.
      • et al.
      Vitamin D as an immune modulator in multiple sclerosis, a review.
      ;
      • Smolders J.
      • et al.
      An update on vitamin D and disease activity in multiple sclerosis.
      ), infection with Epstein-Barr virus (EBV) (
      • Ascherio A.
      • Munger K.L.
      Epidemiology of multiple sclerosis: from risk factors to prevention-an update.
      ;
      • Bar-Or A.
      • et al.
      Epstein-Barr virus in multiple sclerosis: theory and emerging immunotherapies.
      ;
      • Kvistad S.
      • et al.
      Antibodies to Epstein-Barr virus and MRI disease activity in multiple sclerosis.
      ), smoking (
      • Arneth B.
      Multiple sclerosis and smoking.
      ) and obesity (
      • Ascherio A.
      • Munger K.L.
      Epidemiology of multiple sclerosis: from risk factors to prevention-an update.
      ). Especially EBV, a B cell-tropic virus that remains latent in memory B populations, appears to be a prerequisite for developing MS. (
      • Abrahamyan S.
      • et al.
      Complete Epstein-Barr virus seropositivity in a large cohort of patients with early multiple sclerosis.
      ;
      • Pakpoor J.
      • et al.
      The risk of developing multiple sclerosis in individuals seronegative for Epstein-Barr virus: a meta-analysis.
      ;
      • Tselis A.
      Epstein-Barr virus cause of multiple sclerosis.
      ) Circulating antibodies against the EBV nuclear antigen type 1 (EBNA-1) are also associated with a higher risk of radiological MS activity (
      • Kvistad S.
      • et al.
      Antibodies to Epstein-Barr virus and MRI disease activity in multiple sclerosis.
      ).
      Of the lymphocyte populations, B cells have recently gained a lot of attention, largely due to the positive effects of B cell depleting CD20-targeted therapies on MS disease activity in both relapsing and progressive MS. (
      • Gelfand J.M.
      • Cree B.A.C.
      • Hauser S.L.
      Ocrelizumab and other CD20(+) B-cell-depleting therapies in multiple sclerosis.
      ;
      • Hauser S.L.
      • et al.
      Ocrelizumab versus interferon beta-1a in relapsing multiple sclerosis.
      ) Where B cells were traditionally viewed as a relatively passive population in the older MS disease models (
      • Hemmer B.
      • et al.
      Pathogenesis of multiple sclerosis: an update on immunology.
      ), more recent models give them a more central role (
      • Wanleenuwat P.
      • Iwanowski P.
      Role of B cells and antibodies in multiple sclerosis.
      ;
      • Milo R.
      Therapies for multiple sclerosis targeting B cells.
      ;
      • Probstel A.K.
      • Hauser S.L.
      Multiple sclerosis: B cells take center stage.
      ), partially due to the growing appreciation for their antibody-independent functions in (auto)immunity (
      • Li R.
      • Patterson K.R.
      • Bar-Or A.
      Reassessing B cell contributions in multiple sclerosis.
      ). This includes their role as antigen presenting cell (APC), their production of pro-inflammatory cytokines, as well as their involvement in establishing and maintaining meningeal tertiary follicles, as seen in secondary progressive MS patients (
      • Serafini B.
      • et al.
      Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis.
      ). These tertiary follicles in the secondary progressive (SP)MS phase are associated with an earlier onset of disease, as well as a more severe disease course (
      • Magliozzi R.
      • et al.
      Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology.
      ). Since EBV primarily infects (memory) B cells (
      • Bar-Or A.
      • et al.
      Epstein-Barr virus in multiple sclerosis: theory and emerging immunotherapies.
      ;
      • Laurence M.
      • Benito-Leon J.
      Epstein-Barr virus and multiple sclerosis: updating Pender’s hypothesis.
      ) and EBV infected B cells have been found in the tertiary follicles by some (
      • Magliozzi R.
      • et al.
      B-cell enrichment and Epstein-Barr virus infection in inflammatory cortical lesions in secondary progressive multiple sclerosis.
      ), but not all groups (
      • Peferoen L.A.
      • et al.
      Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
      ;
      • Willis S.N.
      • et al.
      Epstein-Barr virus infection is not a characteristic feature of multiple sclerosis brain.
      ), it is suggestive that these factors are closely linked in the pathogenesis of MS.
      Besides treatment of existing clinical manifestations, predicting subsequent disease activity is a challenge in MS. Accurate prediction would facilitate timely intervention, thereby reducing lesions and limiting permanent damage to the CNS. Recently, we showed that supplementation of vitamin D3 for 48 weeks in interferon-beta-1a treated relapsing-remitting (RR)MS patients did not increase the proportion reaching no evidence of disease activity (NEDA), but was associated with a reduced proportion of combined unique active (CUA) lesions on week 48 MRI (i.e. new T2 or gadolinium-enhancing lesions) (
      • Hupperts R.
      • et al.
      Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
      ). In N = 50 participants of a Dutch sub-study of this trial, we found that a low ratio between NK cells and IL-17A producing T helper cells (NK/IL-17A+CD4+ cell ratio) at week 0 predicted the presence of CUA at 48 weeks (
      • Mimpen M.
      • et al.
      Prognostic value of natural killer cell/T cell ratios for disease activity in multiple sclerosis.
      ). Moreover, this ratio was associated with IL-2 receptor alpha chain expression and shedding (
      • Mimpen M.
      • et al.
      NK/T cell ratios associate with interleukin-2 receptor alpha chain expression and shedding in multiple sclerosis.
      ).
      We now assess the prognostic value of circulating B cell phenotypes in the same cohort for MRI activity after 48 weeks. Furthermore, we explore how these phenotypes associate with anti-EBV serology, i.e. anti-EBNA-1 IgG and anti-VCA IgG, and other lymphocyte subsets.

      2. Methods

      2.1 Patients

      This study is a post-hoc extended analysis of the SOLARIUM study, which was a sub-study of the SOLAR study (NCT01285401). The aim of the SOLAR study was to evaluate disease activity in interferon beta-1a (IFN-β-1a) treated RRMS patients using high dose vitamin D3 supplements versus placebo. Patients in the vitamin D3 group received cholecalciferol drops (Vigantol Oil, Merck) 7000IU/day in the first 4weeks, followed by 14,000IU/day up to week 48. The SOLARIUM sub-study investigated the effect of high dose vitamin D3 supplementations on immune system composition. In- and exclusion criteria for the SOLAR and SOLARIUM studies are described elsewhere (
      • Hupperts R.
      • et al.
      Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
      ;
      • Muris A.H.
      • et al.
      Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNbeta; the SOLARIUM study.
      ). In short, the SOLAR study recruited patients aged 18–55 years, diagnosed with RRMS (according to the McDonald criteria 2005) confirmed by typical MS findings on magnetic resonance imaging (MRI). The first clinical event had to be described within 5 years prior to study screening and signs of active disease must have been present in the last 18 months, but no relapse in 30 days before inclusion. Patients could not participate if they already consumed more than 1000 IU (25 μg) of vitamin D3 supplements. All patients received IFN-β-1a 44 μg s.c. three times weekly. Eligible participants had used IFN-β-1a at least 90 days, but no longer than 18 months. After randomisation, the patients received either IFN-β-1a and a placebo or IFN-β-1a and vitamin D3 supplements.
      Regarding the MRI outcome, procedures and findings as reported in the SOLAR trial were used (
      • Hupperts R.
      • et al.
      Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
      ). In short, MRI assessments were performed at baseline and week 48. Scans included T2- and T1-weighted images (3 mm slice thickness and 1 mm in-plane resolution) before and after administration of IV gadolinium. MRI was used to find presence of combined unique active (CUA) lesions (new gadolinium-enhancing or new/enlarging T2 lesions) at week 48. Since only N = 3 patients had more than 1 CUA after 48 weeks, MRI activity was measured as a dichotomous yes/no outcome.
      The SOLARIUM sub-study recruited patients from four of the five participating centers in the Netherlands without adding additional in- or exclusion criteria, being eligible when they consented to participation in the sub-study. Written informed consent was acquired and the SOLARIUM study was approved by the Ethical Committee METC-Z (11-T-03; Heerlen, the Netherlands). Peripheral blood samples were collected at baseline (w0) and after 48 weeks (w48) and analysed using flow cytometry.
      For the current study, N = 50 participants of whom data regarding B cells and EBV antibody parameters were available could be included.

      2.2 Peripheral blood mononuclear cells isolation

      The acquirement and analysis of the peripheral blood mononuclear cells (PBMCs) is described elsewhere (
      • Muris A.H.
      • et al.
      Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNbeta; the SOLARIUM study.
      ). In summary, peripheral blood samples were collected from patients at baseline and week 48 of treatment. Blood was collected in a 10 mL sodium heparin blood sampling tube (BD Biosciences, Breda, The Netherlands) and transported to Maastricht University Medical Center, the Netherlands, at room temperature. Within 24 h PBMCs were isolated by gradient centrifugation as described in previous publications (
      • Hupperts R.
      • et al.
      Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
      ;
      • Muris A.H.
      • et al.
      Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNbeta; the SOLARIUM study.
      ).

      2.3 Flow cytometry

      Immediately after isolation, PBMCs were stained with a cocktail of monoclonal antibodies in order to define B cells (CD19+ lymphocytes) and subsequently transitional B cells (IgD+CD27CD38++), naïve B cells (IgD+CD27CD38+), non-isotype switched B cells (IgD+CD27+CD38+/−), isotype switched B cells (IgDCD27+CD38+/−), plasmablasts (IgDCD38++) and senescent B cells (IgDCD27). The following fluorochrome-conjugated antibodies were used: IgD-FITC (BD Biosciences, Breda, The Netherlands); CD27-PE (BD Biosciences); CD19-PerCP-Cy5–5 (BD Biosciences); CD38-APC (BD Biosciences). Additionally, regulatory B cells (Bregs, CD19+IL-10+) were defined by IL-10 production upon stimulation with CpG, as described in an earlier publication (
      • Muris A.H.
      • et al.
      Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNbeta; the SOLARIUM study.
      ). For FACS analysis (FACS Canto II flow cytometer (BD Biosciences)), B cells were analysed for 100,000 events in the lymphocyte gate. FACS DIVA software (BD Biosciences) was used to analyse the flow cytometry data. Gating strategies, as well as phenotype definitions, are shown in Fig. 1. The definition of transitional B cells and plasmablasts was validated in a subset of participants at week 48 using an alternative gating strategy (CD19+CD24++CD38++ and CD19+CD27++CD38++, respectively; Supplementary Fig. 1).
      Fig. 1
      Fig. 1Gating strategy used to analyse and define B cells and subsets. Step 1 shows the gating of lymphocytes from the PBMC sample. Step 2 shows the gating of B cells, defined as CD19+ lymphocytes. Step 3 and 4 show distinctions made based on the IgD, CD27 and CD38 marker. Based on this strategy, six B cell phenotypes can be defined: transitional (IgD+CD38++), naïve (IgD+CD27CD38+), non-isotype switched (IgD+CD27+CD38+/−), isotype switched (IgDCD27+CD38+/−), plasmablasts (IgDCD38++) and senescent (IgDCD27). APC: allophycocyanin; FITC: fluorescein isothiocyanate; FSC: forward scatter; PE: phycoerythrin SSC: side scatter; PerCP: peridinin chlorophyll protein complex.

      2.4 Antibody measurements

      From SOLARIUM participants, blood was drawn at baseline and after a 48-week study period for measurements of several markers. Levels of IgG against the EBV antigens EBNA-1 and viral capsid antigen (VCA) were measured in plasma samples, which were stored at −20 °C until analyses. Tests were performed using the quantitative LIAISON® EBNA or VCA IgG assays (DiaSorin, Saluggia, Italy), which use chemiluminescence immunoassay technology. Results >22U/mL were considered positive.

      2.5 Statistics

      SPSS software (IBM SPSS, version 25.0. Chicago, IL) was used to assess associations with disease activity. Normality of data was assessed by visual inspection of histograms with normal curves, skewness and kurtosis. To assess the association between B cell phenotypes and MRI activity after 48 weeks, independent t-tests or Mann-Whitney U tests were performed based on distribution of data. When assessing the role of multiple parameters for MRI activity after 48 weeks, a binary logistic model was used. If any parameters were not normally distributed according to our earlier mentioned criteria, they were log-transformed in order to normalise the data.
      To determine cut-off points for an exploratory multi-factor model, ROC curves were plotted. The cut-off with the highest combined sensitivity and specificity was used. A p-value of <0.05 was considered statistically significant.

      3. Results

      3.1 Baseline characteristics

      The SOLARIUM cohort consisted of 53 RRMS patients, of which N = 3 patients were ineligible due to incomplete immunostainings. Additionally, N = 3 patients did not undergo an MRI examination after 48 weeks, leaving N = 47 patients for analyses regarding MRI outcome. N = 11 patients were positive for presence of CUA after 48 weeks follow-up, whereas N = 36 were negative. Baseline characteristics did not differ between patients with and without MRI activity, except for a larger proportion placebo-randomized patients in the group with MRI activity (Supplementary Table 1) (
      • Mimpen M.
      • et al.
      Prognostic value of natural killer cell/T cell ratios for disease activity in multiple sclerosis.
      ;
      • Hupperts R.
      • et al.
      Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
      ). As reported earlier, all participants were EBV-seropositive: 92% of patients were anti-EBNA-1 positive while 96% were positive for anti-VCA, and none were negative for both markers (
      • Rolf L.
      • et al.
      Exploring the effect of vitamin D3 supplementation on the anti-EBV antibody response in relapsing-remitting multiple sclerosis.
      ).

      3.2 B cell compartment composition associates with MRI activity after 48 weeks

      First, we assessed the association between circulating B cell subsets and the presence of CUA on week 48 MRI. At week 0, a lower proportion of transitional B cells [p = 0.004] and, to a lesser extent, a higher proportion of isotype switched B cells [p = 0.030] were found in patients with CUA on the week 48 MRI (Fig. 2A ). At week 48, similar associations were observed for transitional and isotype switched B cells [p = 0.002 and p = 0.015, respectively] (Fig. 2B) with the addition of a higher percentage of non-isotype switched B-cells in participants with CUA on the week 48 MRI [p = 0.035]. These data suggest higher proportions of transitional B cells, and to a lesser extent also lower proportions of isotype-switched B cells, to positively influence the risk for CUA in interferon beta-treated RRMS. Although our manuscript is focused on MRI activity of MS, as being the most sensitive marker for inflammatory disease activity for MS (
      • Barkhof F.
      • et al.
      MRI monitoring of immunomodulation in relapse-onset multiple sclerosis trials.
      ), we also explored an association of B cells subsets with the absence of the clinical and MRI activity as expressed in the composite NEDA-3 endpoint. Patients with a NEDA-3 status at 48 week follow-up showed higher proportions of transitional B cells at baseline (p = 0.008, Supplementary Fig. 2). To exclude an effect of vitamin D supplementation on circulating B cell subsets, we explored effects of vitamin D supplementation on these phenotypes. None of the B cell subsets showed a significant change due to vitamin D3 supplements (Fig. 2C).
      Fig. 2
      Fig. 2A: Differences in B cell phenotypes (as defined in , with the addition of regulatory B cells) between patients with and without MRI activity, measured at baseline. Shown p-value is calculated using a Mann-Whitney U test. B: Differences in B cell phenotypes (as defined in , with the addition of regulatory B cells) between patients with and without MRI activity, measured at week 48. Shown p-value is calculated using a Mann-Whitney U test. C: Differences in the evolution of B cells over 48 weeks between patients with and without vitamin D supplementation. Evolution is calculated as the log ratio of week 48/w0, where negative numbers represent a relative decrease of B cell phenotypes, while positive numbers indicate a relative increase in B cell phenotypes. Shown p-value is calculated using a Mann-Whitney U test. Dotted lines represent the 0 value.

      3.3 B cell subsets do not associate with anti-EBNA-1 IgG levels in serum

      Since circulating antibodies against EBV antigens have been reported to be associated with MS MRI activity (
      • Kvistad S.
      • et al.
      Antibodies to Epstein-Barr virus and MRI disease activity in multiple sclerosis.
      ), associations between B cell phenotypes and IgG responses against EBNA-1 and VCA were explored. In our cohort, there was no difference in anti-EBNA-1 and anti-VCA antibodies between patients with or without CUA on week 48 MRI (Fig. 3A and B ). Patients without MRI-activity at 48 weeks showed a more pronounced reduction in circulating anti-EBNA-1 but not anti-VCA IgG antibodies during 48 weeks of follow-up compared to patients with CUA (Fig. 3C). Accordingly, vitamin D3 supplementation was already shown to be associated with both a lower proportion of CUA at week 48 (
      • Hupperts R.
      • et al.
      Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
      ), and a reduction of circulating anti-EBNA-1 IgG antibodies (
      • Rolf L.
      • et al.
      Exploring the effect of vitamin D3 supplementation on the anti-EBV antibody response in relapsing-remitting multiple sclerosis.
      ). In our cohort, circulating anti-EBNA-1 IgG antibodies did not correlate with B cell phenotype percentages at any time point (Fig. 3D). This finding suggests that changes in B cell phenotypes and anti-EBNA-1 IgG in serum are independent contributors to CUA-risk in IFN-β-1a treated MS patients.
      Fig. 3
      Fig. 3A: Differences in anti-EBNA-1 IgG and anti-VCA IgG between patients with and without MRI activity, measured at baseline. Shown p-value was calculated using a Mann-Whitney U test. B: Differences in anti-EBNA-1 IgG and anti-VCA IgG between patients with and without MRI activity, measured at week 48C: Differences in anti-EBNA-1 IgG and anti-VCA IgG between patients with and without MRI activity, measured as the difference between week 48 and baseline. D: Heatmap showing correlations between anti-EBV serology and B cell subsets. Only statistically significant correlations are coloured. A green colour indicates a positive correlation. Correlation coefficient shown is a Spearman's rho. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

      3.4 Patients with MRI activity show distinct clustering for multiple prognostic parameters

      Since B cell phenotypes appeared a correlate of neither vitamin D supplementation, nor anti-EBNA-1 IgG levels, we further explored how individual parameters contribute to CUA lesions at week 48. Our previous work showed that the week 0 ratio between NK cells and IL-17A+CD4+ T cells predicted the presence of CUA on week 48 MRI (
      • Mimpen M.
      • et al.
      Prognostic value of natural killer cell/T cell ratios for disease activity in multiple sclerosis.
      ) and as such, this parameter was also included in our model. When introducing these 4 predictors of CUA on week 48 MRI in a 3D plot, patients with MRI activity tended to cluster together (Fig. 4A ). After log-transforming data that were not normally distributed, we introduced our parameters in an explorative binary logistic regression model where they explained 57.9% of the variance in CUA-activity in our cohort (Nagelkerke R2 = 0.579), and all factors except Δ anti-EBNA-1 IgG (Δw48/w0) trended to contribute to this model (Supplementary Table 2). To visualize this interplay for all individual participants, we dichotomized all variables based on individual ROC-curves in a high- and low-risk profile for CUA (Fig. 4B). Indeed, participants with CUA on week 48 MRI-scan tended to show a high-risk profile for multiple predictors.
      Fig. 4
      Fig. 4A: 3D plot using three prognostic markers: relative presence of transitional B cells in the B cell population, NK/IL-17A+CD4+ T cell ratio and the difference in anti-EBNA-1 IgG antibodies between week 48 and baseline. Red markers represent patients with MRI activity after 48 weeks, while black markers represent patients without MRI activity. Triangle markers represent patients who received high dose vitamin D3 supplementation, while round markers represent patients who received placebo. B: Visualisation of multiple-hit model. When grouping patients based on the amount of favourable or unfavourable prognostic markers using cutoffs shown, patients with MRI activity (shown in red) tend to cluster around multiple negative markers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

      4. Discussion

      We investigated the prognostic value of B cell subsets, and explored associations with other predictors of MS MRI activity in a homogenous cohort of interferon-β-1a treated RRMS patients. We show that low circulating proportions of transitional B cells associate with a lower risk of CUA on week 48 MRI, and that this is no correlate of proportions of IL-10+ Breg cells. Additionally, we show that this association of high transitional B cell proportions with a lower risk of CUA is not dependent on vitamin D supplementation, circulating anti-EBNA-1 IgG levels, or the ratio between NK and IL-17A+CD4+ T cells. Altogether, we conclude that the risk of MS MRI activity during interferon beta therapy is likely the result of many interacting phenotypes and effector characteristics of individual lymphocyte populations. Herewith, our data are a call for a systems biology approach to further understand the role of lymphocytes in the disease process of RRMS.
      Our finding that transitional B cells may contribute to a protective effect in RRMS is in line with earlier research. Transitional B cells are shown to have regulatory properties (
      • Zhou Y.
      • et al.
      Transitional B cells involved in autoimmunity and their impact on neuroimmunological diseases.
      ). As such, they have been implied in the pathogenesis of MS. Indeed, reduced transitional B cell numbers (
      • Lee-Chang C.
      • et al.
      Primed status of transitional B cells associated with their presence in the cerebrospinal fluid in early phases of multiple sclerosis.
      ;
      • Miyazaki Y.
      • et al.
      Suppressed pro-inflammatory properties of circulating B cells in patients with multiple sclerosis treated with fingolimod, based on altered proportions of B-cell subpopulations.
      ) and regulatory function (
      • Cencioni M.T.
      • et al.
      Defective CD19+CD24(hi)CD38(hi) transitional B-cell function in patients with relapsing-remitting MS.
      ) and increased migratory capacity (
      • Lee-Chang C.
      • et al.
      Primed status of transitional B cells associated with their presence in the cerebrospinal fluid in early phases of multiple sclerosis.
      ) have been reported in clinically isolated syndrome and MS. Additionally, some MS treatments associated with reduced disease activity in MS have been associated with increased transitional B cell proportions, including fingolimod (
      • Miyazaki Y.
      • et al.
      Suppressed pro-inflammatory properties of circulating B cells in patients with multiple sclerosis treated with fingolimod, based on altered proportions of B-cell subpopulations.
      ;
      • Miyazaki Y.
      • et al.
      Fingolimod induces BAFF and expands circulating transitional B cells without activating memory B cells and plasma cells in multiple sclerosis.
      ;
      • Blumenfeld S.
      • Staun-Ram E.
      • Miller A.
      Fingolimod therapy modulates circulating B cell composition, increases B regulatory subsets and production of IL-10 and TGFbeta in patients with Multiple Sclerosis.
      ) and interferon-β (
      • Dooley J.
      • et al.
      Immunologic profiles of multiple sclerosis treatments reveal shared early B cell alterations.
      ). As such, one should exercise caution to extrapolate our findings to MS patients treated with other disease modifying therapies. Both fingolimod and IFN-β have been reported to increase transitional B cells through the increased circulating levels of B-cell activating factor of the TNF family (BAFF) (
      • Miyazaki Y.
      • et al.
      Fingolimod induces BAFF and expands circulating transitional B cells without activating memory B cells and plasma cells in multiple sclerosis.
      ;
      • Hedegaard C.J.
      • et al.
      Interferon-beta increases systemic BAFF levels in multiple sclerosis without increasing autoantibody production.
      ). However, since BAFF was not significantly affected by high-dose vitamin D3 supplementation in a preceding pilot-study (
      • Knippenberg S.
      • et al.
      Effect of vitamin D(3) supplementation on peripheral B cell differentiation and isotype switching in patients with multiple sclerosis.
      ), we did not include BAFF measurements in the current SOLARIUM study-design.
      In several studies, the protective effect of transitional B cells has been attributed to their capability of producing IL-10 (
      • Miyazaki Y.
      • et al.
      Suppressed pro-inflammatory properties of circulating B cells in patients with multiple sclerosis treated with fingolimod, based on altered proportions of B-cell subpopulations.
      ;
      • Cencioni M.T.
      • et al.
      Defective CD19+CD24(hi)CD38(hi) transitional B-cell function in patients with relapsing-remitting MS.
      ;
      • Blumenfeld S.
      • Staun-Ram E.
      • Miller A.
      Fingolimod therapy modulates circulating B cell composition, increases B regulatory subsets and production of IL-10 and TGFbeta in patients with Multiple Sclerosis.
      ). In our study, an association with MRI-activity after 48 weeks follow-up was found only for transitional B cells, but not for IL-10+ B cells. This interesting contrast may be explained by the method to analyse IL-10+ B cells. In our study, B cells were stimulated with CpG for 24 h, after which IL-10+ B cells were gated. One study shows that a combination of CpG and CD40L stimulation mainly expands regulatory B cells with a memory phenotype (
      • Banko Z.
      • et al.
      Induction and differentiation of IL-10-producing regulatory B cells from healthy blood donors and rheumatoid arthritis patients.
      ). As such, it may be that transitional B cells exert their protective effect through a Breg phenotype, but that the transitional Breg population remains undetected by our induction method. Alternatively, transitional B cells may control CD4+ T cell proliferation and MS disease activity by other effector mechanisms than IL-10 (
      • Simon Q.
      • et al.
      In-depth characterization of CD24(high)CD38(high) transitional human B cells reveals different regulatory profiles.
      ).
      Isotype-switched memory B cells are generally being viewed as an unfavourable cell subset in MS. (
      • Dooley J.
      • et al.
      Immunologic profiles of multiple sclerosis treatments reveal shared early B cell alterations.
      ) Our current results are in line with these notion, as higher percentages of isotype switched B cells are associated with a higher risk of CUA presence. The lack of correlation between anti-EBNA-1 IgG and isotype switched B cells is interesting, as EBV is known for its latent infection of memory B cells. One possible explanation may lie in recent evidence, suggesting that memory B cells only develop into antigen secreting cells after migrating to the CNS (
      • van Langelaar J.
      • et al.
      The association of Epstein-Barr virus infection with CXCR3(+) B-cell development in multiple sclerosis: impact of immunotherapies.
      ). Isotype switched B cells as measured in peripheral blood may, therefore, give an incomplete view of anti-EBNA-1 production.
      We showed that transitional B cell frequencies associate with MS CUA presence, as also observed for other immune-related predictors identified earlier in this cohort, including anti-EBNA-1 IgG (
      • Rolf L.
      • et al.
      Exploring the effect of vitamin D3 supplementation on the anti-EBV antibody response in relapsing-remitting multiple sclerosis.
      ), vitamin D supplementation (
      • Hupperts R.
      • et al.
      Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
      ), and NK/T cell ratios (
      • Mimpen M.
      • et al.
      Prognostic value of natural killer cell/T cell ratios for disease activity in multiple sclerosis.
      ). This observation fits the growing appreciation for the complex interplay between environmental factors and genetic background that influences cell subsets in (auto)immune diseases (
      • Brodin P.
      • Davis M.M.
      Human immune system variation.
      ;
      • Davis M.M.
      • Tato C.M.
      • Furman D.
      Systems immunology: just getting started.
      ;
      • Ma’ayan A.
      Complex systems biology.
      ). As such, the need for integrative models has increased, which led to an increase in popularity for a systems biology approach, where larger interacting systems are taken into account. For MS, a systems biology approach has been used to identify e.g. regulatory genetic pathways (
      • International Multiple Sclerosis Genetics, C
      A systems biology approach uncovers cell-specific gene regulatory effects of genetic associations in multiple sclerosis.
      ) as well as potential biomarkers for disease activity (
      • Chase Huizar C.
      • Raphael I.
      • Forsthuber T.G.
      Genomic, proteomic, and systems biology approaches in biomarker discovery for multiple sclerosis.
      ). An approach on a cellular level remains poorly investigated. In our exploratory model, the combination of several prognostic factors, mainly the relatively increased or decreased presence of transitional B cells, NK cells and IL-17A producing T helper cells, leads to a model which currently explains over half of the variance in the prognosis of CUA in IFN-β-1a treated RRMS patients. To make more definitive claims on this approach, more biomarkers should be investigated and integrated into a larger model using a larger dataset. Nonetheless, our findings underline the importance of looking at the interaction rather than merely the presence of individual immune components to understand the complex pathogenesis of diseases like MS.
      Our study has a some limitations. First, our data is derived from the SOLAR and SOLARIUM studies, which both were designed to answer a different research question. Thus, the exploratory nature of our research brings an increased risk for false negatives, in addition to the absence of a few potentially relevant parameters like the aforementioned BAFF levels. Additionally, our patients exclusively used interferon-β-1a. While this greatly increased homogenisation of the participants, it may influence the extrapolation of our data. Strengths of this study include its double-blinded nature, as well as its broad view of the B cell compartment.
      In conclusion, our data underline the protective role for transitional B cells in IFN-β treated RRMS patients, as well as its potential as a prognostic biomarker for MRI activity. As noted earlier, our data suggests an interplay between several prognostic factors and calls for a systems biology approach to further grasp the interactions of lymphocyte subsets in RRMS. More research is necessary to confirm the prognostic value of transitional B cells in treatment-naïve RRMS patients and also to investigate other potential biomarkers in a systems biology approach.

      Data availability statement

      The data presented in this study are available from the corresponding author upon reasonable request.

      Declaration of Competing Interest

      MM has nothing to disclose; JD has nothing to disclose; LR has nothing to disclose; AHM has nothing to disclose; WD has nothing to disclose; RH received institutional research grants and fees for lectures and advisory boards from Biogen, Merck, and Genzyme-Sanofi; ML has nothing to disclose; OG has nothing to disclose; JS received lecture and/or consultancy fees of Biogen, Merck, Sanofi-Genzyme, and Novartis.

      Acknowledgments

      This study was funded by Nationaal MS Fonds grant OZ2016-001 and an unrestricted grant by Merck.

      Appendix A. Supplementary data

      References

        • Abrahamyan S.
        • et al.
        Complete Epstein-Barr virus seropositivity in a large cohort of patients with early multiple sclerosis.
        J. Neurol. Neurosurg. Psychiatry. 2020; 91: 681-686
        • Arneth B.M.
        Impact of B cells to the pathophysiology of multiple sclerosis.
        J. Neuroinflammation. 2019; 16: 128
        • Arneth B.
        Multiple sclerosis and smoking.
        Am. J. Med. 2020; 133: 783-788
        • Ascherio A.
        • Munger K.L.
        Epidemiology of multiple sclerosis: from risk factors to prevention-an update.
        Semin. Neurol. 2016; 36: 103-114
        • Banko Z.
        • et al.
        Induction and differentiation of IL-10-producing regulatory B cells from healthy blood donors and rheumatoid arthritis patients.
        J. Immunol. 2017; 198: 1512-1520
        • Barkhof F.
        • et al.
        MRI monitoring of immunomodulation in relapse-onset multiple sclerosis trials.
        Nat. Rev. Neurol. 2011; 8: 13-21
        • Bar-Or A.
        • et al.
        Epstein-Barr virus in multiple sclerosis: theory and emerging immunotherapies.
        Trends Mol. Med. 2020; 26: 296-310
        • Blumenfeld S.
        • Staun-Ram E.
        • Miller A.
        Fingolimod therapy modulates circulating B cell composition, increases B regulatory subsets and production of IL-10 and TGFbeta in patients with Multiple Sclerosis.
        J. Autoimmun. 2016; 70: 40-51
        • Brodin P.
        • Davis M.M.
        Human immune system variation.
        Nat. Rev. Immunol. 2017; 17: 21-29
        • Cencioni M.T.
        • et al.
        Defective CD19+CD24(hi)CD38(hi) transitional B-cell function in patients with relapsing-remitting MS.
        Mult. Scler. 2020; 27: 1187-1197
        • Chase Huizar C.
        • Raphael I.
        • Forsthuber T.G.
        Genomic, proteomic, and systems biology approaches in biomarker discovery for multiple sclerosis.
        Cell. Immunol. 2020; 358: 104219
        • Davis M.M.
        • Tato C.M.
        • Furman D.
        Systems immunology: just getting started.
        Nat. Immunol. 2017; 18: 725-732
        • Dobson R.
        • Giovannoni G.
        Multiple sclerosis - a review.
        Eur. J. Neurol. 2019; 26: 27-40
        • Dooley J.
        • et al.
        Immunologic profiles of multiple sclerosis treatments reveal shared early B cell alterations.
        Neurol. Neuroimmunol. Neuroinflamm. 2016; 3e240
        • Durelli L.
        • et al.
        T-helper 17 cells expand in multiple sclerosis and are inhibited by interferon-beta.
        Ann. Neurol. 2009; 65: 499-509
        • Gelfand J.M.
        • Cree B.A.C.
        • Hauser S.L.
        Ocrelizumab and other CD20(+) B-cell-depleting therapies in multiple sclerosis.
        Neurotherapeutics. 2017; 14: 835-841
        • Gutcher I.
        • Becher B.
        APC-derived cytokines and T cell polarization in autoimmune inflammation.
        J. Clin. Invest. 2007; 117: 1119-1127
        • Hauser S.L.
        • et al.
        Ocrelizumab versus interferon beta-1a in relapsing multiple sclerosis.
        N. Engl. J. Med. 2017; 376: 221-234
        • Hedegaard C.J.
        • et al.
        Interferon-beta increases systemic BAFF levels in multiple sclerosis without increasing autoantibody production.
        Mult. Scler. 2011; 17: 567-577
        • Hemmer B.
        • et al.
        Pathogenesis of multiple sclerosis: an update on immunology.
        Curr. Opin. Neurol. 2002; 15: 227-231
        • Hupperts R.
        • et al.
        Randomized trial of daily high-dose vitamin D3 in patients with RRMS receiving subcutaneous interferon beta-1a.
        Neurology. 2019; 93: e1906-e1916
        • International Multiple Sclerosis Genetics, C
        A systems biology approach uncovers cell-specific gene regulatory effects of genetic associations in multiple sclerosis.
        Nat. Commun. 2019; 10: 2236
        • Knippenberg S.
        • et al.
        Effect of vitamin D(3) supplementation on peripheral B cell differentiation and isotype switching in patients with multiple sclerosis.
        Mult. Scler. 2011; 17: 1418-1423
        • Kvistad S.
        • et al.
        Antibodies to Epstein-Barr virus and MRI disease activity in multiple sclerosis.
        Mult. Scler. 2014; 20: 1833-1840
        • Laurence M.
        • Benito-Leon J.
        Epstein-Barr virus and multiple sclerosis: updating Pender’s hypothesis.
        Mult. Scler. Relat. Disord. 2017; 16: 8-14
        • Lee-Chang C.
        • et al.
        Primed status of transitional B cells associated with their presence in the cerebrospinal fluid in early phases of multiple sclerosis.
        Clin. Immunol. 2011; 139: 12-20
        • Li R.
        • Patterson K.R.
        • Bar-Or A.
        Reassessing B cell contributions in multiple sclerosis.
        Nat. Immunol. 2018; 19: 696-707
        • Ma’ayan A.
        Complex systems biology.
        J. R. Soc. Interface. 2017; : 14(134)
        • Magliozzi R.
        • et al.
        Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology.
        Brain. 2007; 130: 1089-1104
        • Magliozzi R.
        • et al.
        B-cell enrichment and Epstein-Barr virus infection in inflammatory cortical lesions in secondary progressive multiple sclerosis.
        J. Neuropathol. Exp. Neurol. 2013; 72: 29-41
        • McKinney E.F.
        • et al.
        A CD8(+) NK cell transcriptomic signature associated with clinical outcome in relapsing remitting multiple sclerosis.
        Nat. Commun. 2021; 12: 635
        • Milo R.
        Therapies for multiple sclerosis targeting B cells.
        Croat. Med. J. 2019; 60: 87-98
        • Mimpen M.
        • et al.
        Prognostic value of natural killer cell/T cell ratios for disease activity in multiple sclerosis.
        Eur. J. Neurol. 2020; 28: 901-909
        • Mimpen M.
        • et al.
        Natural killer cells in multiple sclerosis: a review.
        Immunol. Lett. 2020; 222: 1-11
        • Mimpen M.
        • et al.
        NK/T cell ratios associate with interleukin-2 receptor alpha chain expression and shedding in multiple sclerosis.
        J. Neuroimmunol. 2021; 353: 577499
        • Miyazaki Y.
        • et al.
        Suppressed pro-inflammatory properties of circulating B cells in patients with multiple sclerosis treated with fingolimod, based on altered proportions of B-cell subpopulations.
        Clin. Immunol. 2014; 151: 127-135
        • Miyazaki Y.
        • et al.
        Fingolimod induces BAFF and expands circulating transitional B cells without activating memory B cells and plasma cells in multiple sclerosis.
        Clin. Immunol. 2018; 187: 95-101
        • Muris A.H.
        • et al.
        Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNbeta; the SOLARIUM study.
        J. Neuroimmunol. 2016; 300: 47-56
        • Pakpoor J.
        • et al.
        The risk of developing multiple sclerosis in individuals seronegative for Epstein-Barr virus: a meta-analysis.
        Mult. Scler. 2013; 19: 162-166
        • Peferoen L.A.
        • et al.
        Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
        Brain. 2010; 133e137
        • Pierrot-Deseilligny C.
        • Souberbielle J.C.
        Vitamin D and multiple sclerosis: an update.
        Mult. Scler. Relat. Disord. 2017; 14: 35-45
        • Probstel A.K.
        • Hauser S.L.
        Multiple sclerosis: B cells take center stage.
        J. Neuroophthalmol. 2018; 38: 251-258
        • Rolf L.
        • et al.
        Exploring the effect of vitamin D3 supplementation on the anti-EBV antibody response in relapsing-remitting multiple sclerosis.
        Mult. Scler. 2018; 24: 1280-1287
        • Serafini B.
        • et al.
        Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis.
        Brain Pathol. 2004; 14: 164-174
        • Simon Q.
        • et al.
        In-depth characterization of CD24(high)CD38(high) transitional human B cells reveals different regulatory profiles.
        J. Allergy Clin. Immunol. 2016; 137 (e10): 1577-1584
        • Smolders J.
        • et al.
        Vitamin D as an immune modulator in multiple sclerosis, a review.
        J. Neuroimmunol. 2008; 194: 7-17
        • Smolders J.
        • et al.
        An update on vitamin D and disease activity in multiple sclerosis.
        CNS Drugs. 2019; 33: 1187-1199
        • Tselis A.
        Epstein-Barr virus cause of multiple sclerosis.
        Curr. Opin. Rheumatol. 2012; 24: 424-428
        • van Langelaar J.
        • et al.
        T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.
        Brain. 2018; 141: 1334-1349
        • van Langelaar J.
        • et al.
        The association of Epstein-Barr virus infection with CXCR3(+) B-cell development in multiple sclerosis: impact of immunotherapies.
        Eur. J. Immunol. 2021; 51: 626-633
        • Wanleenuwat P.
        • Iwanowski P.
        Role of B cells and antibodies in multiple sclerosis.
        Mult. Scler. Relat. Disord. 2019; 36: 101416
        • Willis S.N.
        • et al.
        Epstein-Barr virus infection is not a characteristic feature of multiple sclerosis brain.
        Brain. 2009; 132: 3318-3328
        • Zhou Y.
        • et al.
        Transitional B cells involved in autoimmunity and their impact on neuroimmunological diseases.
        J. Transl. Med. 2020; 18: 131