Introduction
In patients with multiple sclerosis (MS), anti-CD20 antibody-mediated depletion of B cells enables an excellent therapeutic control of acute relapses [
1,
15,
29]. It is assumed that anti-CD20´s mode of action includes abrogation of both antigen presentation and provision of pro-inflammatory cytokines by B cells [
19], which results in reduced T cell activation. However, besides the development of relapses, most people with MS accumulate permanent disability over time by an underlying, smouldering process within the central nervous system (CNS). This process is not fully understood, but likely initiates relatively early and subtly in the disease course, when acute relapses are clinically more apparent and surfaces as chronic progression when the parenchymal and functional reserve of the CNS is exhausted. Pathophysiologically, chronic progression is attributed to a CNS-intrinsic interplay of CNS-established lymphocytes including T cells, B cells, monocytes, and macrophages within meningeal areas and chronic activation of CNS-resident cells such as microglia und astrocytes in the parenchyma [
6,
18]. What the exact functional role of B and plasma cells is in this process is unclear. In MS, B cells are found in the parenchyma, but primarily in the cerebrospinal fluid (CSF), perivascular locations and meninges [
5,
16,
23,
25,
38]. Especially during periods of active disease, the CSF of patients with MS contains molecules that strongly fosters B cell recruitment and activity [
7,
27] and is enriched in B and plasma cells [
33,
40]. These cells are the source of intrathecally produced immunoglobulin (Ig)G in more than 95% of all patients with MS [
13,
31], suggesting a continuous presence and activation of B cells within the CNS. Hence, it is not surprising that many studies link accelerated disease progression to the presence of B cells in the CNS compartment [
4,
24,
26]. However, besides pro-inflammatory properties, B and plasma cells exhibit various regulatory features [
10]. In the context of experimental CNS autoimmunity both physical interactions [
34,
37,
42] as well as the secretion of anti-inflammatory cytokines e.g. interleukin (IL)-10 have been shown to be relevant for disease regulation [
2,
11,
30,
35,
39] and IL-10-expressing plasma cells have been found in CNS lesions of patients with MS [
25,
32] and mice with experimental autoimmune encephalomyelitis (EAE) [
28]. Moreover, B cells are assumed to control pro-inflammatory activity of other peripheral immune cells [
12,
20,
30], even though IL-10-secreting B cells have been reported to be decreased in number [
17] and impaired in function [
8] in the blood of people with MS. Accordingly, B cells most likely exert both pro- and anti-inflammatory properties in MS. Based on this scenario, and the relatively uncritical use of anti-CD20 mediated pan B cell depletion in MS, but also numerous other chronic inflammatory conditions, we set out to study the functional and possibly clinical impact of B cell regulation in MS and its animal model EAE.
Material and methods
Human samples
After written informed consent was obtained, healthy donors and patients with clinically isolated syndrome, relapsing–remitting or secondary progressive MS were enrolled for our study at the Technical University of Munich, Germany and the University Medical Centre Göttingen, Germany. The study protocol was approved by the ethics committees of the University Medical Centre Göttingen (#03/04/14 and #12/6/21) and the Technical University of Munich (#19/09/10). For detailed characteristics of the study participants, please see Supplementary Tables 1 and 2 (online resource). Patients designated for anti-CD20 antibody treatment had received no corticosteroids or any immunosuppressive or immunomodulatory therapy (other than Rituximab) within two months prior to enrolment. Rituximab was administered at a dose of 1000 mg on days 1 and 15. Blood samples were collected during routine clinical assessment at two time points: before anti-CD20 antibody treatment and 11–28 weeks thereafter when B cells were still absent (confirmed by flow cytometry). Detailed descriptions of the study materials and methods are provided in the sections below.
Mice
Wild-type C57BL/6J mice were purchased from Charles River (Sulzfeld, Germany). MOG p35-55 TCR transgenic 2D2 mice were kindly provided by Dr. Kuchroo (Boston, USA). CD20 KO mice were generated and provided by Genentech. IL-10 KO mice were purchased from Jackson Laboratory. All animal experiments were carried out in accordance with the guidelines of the Central Department for Animal Experiments, University Medical Centre, Göttingen and approved by the Office for Consumer Protection and Food Safety of the State of Lower Saxony (protocol number 33.9-42502-04-15/1804, 33.9-42502-04-19/3244 and 33.9-42502-04-20/3489).
Isolation of human and murine leukocytes
Peripheral blood mononuclear cells (PBMC) from human study subjects were isolated via Ficoll gradient centrifugation. Human total B cells (purity > 98%) and CD27-negative and CD27-positive B cells were isolated from PBMC using the B cell Isolation Kit II and the Memory B cell Isolation Kit (both Miltenyi Biotec), respectively. Human CD14-positive cells were isolated from PBMC by magnetic-activated cell sorting (MACS) using anti-CD14 MicroBeads (Miltenyi Biotec; purity > 90%). Single cell suspensions of murine lymphoid tissues were generated by gentle dissection and passing through 70 µm cell strainer (Greiner Bio-One). Brain and spinal cord tissues were isolated from mice upon perfusion with phosphate-buffered saline (PBS) and were dissociated to single cells using the Multi Tissue Dissociation Kit (Miltenyi Biotec). Murine B cells (purity > 95%) were isolated from spleens by MACS using anti-CD19 MicroBeads (Miltenyi Biotec).
In vitro culture of human B cells
3.5 × 105 MACS-purified B cells of patients with MS and healthy controls, as well as CD27-positive and CD27-negative B cells from healthy individuals, were plated in 96-well u-bottom plates and stimulated with either 4 µg/ml CpG or with 40 µg/ml affiniPure F(ab')2 fragment rabbit anti-human IgM (Fc5 Fragment Specific, Jackson Immuno Research) and 1 µg/ml recombinant human CD40L (R&Dsystem). Unstimulated samples served as controls. After 48 h of culture, supernatants were collected, and secreted IL-6 and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA).
Enzyme-linked immuno spot assay (ELISpot) for analysis of TNF-producing human myeloid cells
3,000 MACS-isolated CD14-positive cells/well were plated in triplicates in TNF capture antibody-coated (human TNFα ELISPOT Antibody Pair, Millipore) Multi-Screen Filter Plates (Millipore) and stimulated with lipopolysaccharide (LPS; E. coli O111:B4; Sigma) for 18 h. Plates were washed and incubated successively with TNF detection antibody, streptavidin–alkaline phosphatase and BCIP/NBT substrate (all human TNF-α ELISPOT Antibody Pair, Millipore). Plates were analysed with an automated imaging system and software (AID ELISpot reader and software, Autoimmun Diagnostika or AELVIS ELISpot reader and software, Stefan Badur Electronic GmbH & Co. KG).
Generation of bone marrow‑derived myeloid cells
To generate bone marrow-derived myeloid cells (BMDM), bone marrow isolated from hind limbs of C57Bl/6J mice was cultured at 37 °C and 5% CO2 for seven days in medium containing 30% conditioned L929 cell supernatant (DMEM, 30% L929 supernatant, 10% foetal calf serum, 5% horse serum, 50 U/ml penicillin, 50 μg/ml streptomycin, 0.05 mM β-mercaptoethanol). Adherent BMDM were detached using 2.5% trypsin (Pan Biotech) and 0.4 mg DNAse I (Roche) and harvested using cell scrapers. Cultures contained > 95% myeloid cells verified by flow cytometry.
Generation of primary microglia
To generate primary microglia, brain cells of new-born to two-day-old C57BL/6J mice were isolated enzymatically using 2.5% trypsin (Pan Biotech) and 0.4 mg DNAse I (Roche). First, a mixed glial cell culture was generated by seeding the cells in DMEM containing 10% foetal calf serum, 1% GlutaMax™, 100 U/ml penicillin, and 100 µg/ml streptomycin and cultivating them at 37° C and 5% CO2 until confluency was reached. To obtain enriched microglia cultures, cells were thereafter stimulated for five days with medium containing 30% conditioned L929 cell supernatant (DMEM, 30% L929 supernatant, 10% foetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin). Primary microglia were harvested by gentle shaking at 90 rpm for 30 min at 37 °C to separate microglial cells from other glia cell. Cultures contained > 97% microglial cells verified by flow cytometry.
Isolation and stimulation of murine B cells, generation of B cell supernatant and neutralization of IL-10
Splenic B cells were isolated from C57Bl/6J mice using MACS (mouse anti-CD19 MicroBeads; BioLegend) and purity (> 95%) was evaluated by flow cytometry. Cytokine secretion was induced by stimulation with 5 µg/ml LPS (E. coli O111:B4; Sigma). For the co-culture experiments, B cells were harvested after 24 h and washed thoroughly to remove LPS. For the experiments with soluble B cell products, B cell supernatants were collected after 48 h of culture and LPS was removed using Pierce™ high capacity endotoxin removal spin columns (ThermoFisher) according to manufacturer’s instructions. Where indicated, IL-10 was neutralized by adding 1 µg/ml anti-IL-10 antibody (clone: JES5-2A5; BioXcell) or isotype control antibody (clone: TNP6A7; BioXcell) to the B cell supernatant 20 min before further use.
Culture of bone marrow-derived myeloid cells and microglia with B cells or their soluble products
BMDM were plated at a density of 0.5 × 105 cells/well into 96-well flat-bottom plates and stimulated with 100 ng/ml LPS. Primary microglia were plated at a density of 2 × 105 cells/well into 12-well plates and stimulated with 1 ng/ml LPS. For co-culture with B cells, 2 × 105 pre-stimulated B cells were added to BMDM or microglia and incubated for 24–48 h. Where indicated, cells were separated by a transwell system (Corning) to inhibit cellular contact between BMDM or microglia and B cells. In brief, BMDM or microglia were cultured on the bottom of a 96-well plate and pre-stimulated B cells were seeded on top into a membrane-insert with 0.4 µm pores that allows exchange of soluble factors, but prevents cellular transmigration. For culture with soluble B cell factors, supernatant of LPS-stimulated B cells, where indicated neutralized for IL-10, was added to BMDM or microglia in a ratio of 1:1. After 24–48 h, cell supernatants were harvested for ELISA, and BMDM/microglia were detached using 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA; w/v) in PBS for analysis by flow cytometry.
Assessment of T cell proliferation and differentiation in vitro
Adherent BMDM or microglia were detached using 0.05% trypsin and 0.02% EDTA (w/v) in PBS. BMDM (0.5 × 105 cells/well) or microglia (0.3 × 105 cells/well) were plated into 96-well flat-bottom plates and stimulated with 100 ng/ml or 1 ng/ml LPS, respectively. Where indicated, rIL-10 (1 ng/ml), complete B cell supernatant or B cell supernatant neutralized for IL-10 were added additionally. After 24 h, BMDM/microglia were washed twice and 0.5 × 105 MACS-purified (Pan T cell Isolation Kit, Miltenyi, Bergisch Gladbach, Germany) carboxyfluorescein succinimidyl ester (CFSE)-stained (CFSE Cell Division Tracker Kit, BioLegend) or unstained T cells from 2D2 mice were added per well. 72 h after co-culture in the presence of MOG p35-55, T cell proliferation and differentiation were evaluated by flow cytometry and/or ELISA.
EAE induction and scoring
As indicated in the respective figure legend, female wild-type mice were immunized subcutaneously with 50 or 75 µg MOG p35-55 MEVGWYRSPFSRVVHLYRNGK (Auspep) or 75 μg MOG protein 1–117 (GenScript Biotech) emulsified in complete Freund’s adjuvant (Sigma-Aldrich) containing 250 µg inactivated Mycobacterium tuberculosis H37 Ra (BD Bioscience) followed by intraperitoneal injections of 200 ng of Bordetella pertussis toxin (Sigma-Aldrich) on the day of immunization and two days thereafter. EAE severity was assessed daily and scored on a scale from 0 to 5 as follows: 0 = no clinical signs; 1.0 = tail paralysis; 2.0 = hindlimb paresis; 3.0 = severe hindlimb paresis; 4.0 = paralysis of both hindlimbs; 4.5 = hindlimb paralysis and beginning forelimb paresis; and 5.0 = moribund/death.
Anti-CD20 treatment
Mice received weekly intraperitoneal injections of 0.2 mg murine monoclonal anti-CD20 antibody (Clone 5D2; IgG2a) or monoclonal anti-HIV-1 (Clone: gp120; IgG2a) control antibody (both provided by Genentech) starting three weeks prior to immunization or at the indicated time points.
Adoptive B cell transfer
C57BL/6J recipient mice were depleted of B cells by weekly intraperitoneal injections of 0.2 mg murine anti-CD20 antibody during the whole experiment. 10 × 106 CD20KO or CD20KO/IL10KO B cells were intravenously injected into recipient mice once a week for three consecutive weeks, starting three weeks after the first anti-CD20 antibody injection. Six weeks after the last B cell transfer, recipient mice were immunized with MOG p35-55 and clinical symptoms were monitored for five weeks.
Detection of anti-MOG antibodies
96-well plates were coated with 10 μg/ml MOG protein 1–117 in PBS overnight. Thereafter, diluted serum samples were incubated for two hours. After washing, plate-bound antibodies were detected with horseradish peroxidase-conjugated anti-mouse IgG, directed against the Fc part of the bound antibodies. Absorbance was measured at 450 nm with subtraction of a 540 nm reference wavelength on the iMark Microplate Reader.
Histology and immunohistochemistry
Mice were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) and tissue was paraffin embedded. One-micrometre thick slices were stained with haematoxylin and eosin and Luxol fast blue/periodic acid-Schiff. T cells, plasma cells, macrophages and microglia were detected by immunohistochemistry with an avidin–biotin technique using antibodies specific for CD3 (SP7; DCS Innovative Diagnostik-Systeme), IgG (polyclonal; Merck), Mac-3 (M3/84; BD Biosciences) and Iba1 (polyclonal; Fujifilm), respectively. Histological sections were captured using a digital camera (DP71; Olympus Europa) mounted on a light microscope (BX51; Olympus Europa). The percentage of demyelinated white matter was calculated using cellSens Dimension software (Olympus Europa). Inflammatory cells were quantified at × 400 magnification using an ocular counting grid and are shown as cells/mm2. At least eight spinal cord cross sections per animal were taken for each analysis.
Enzyme-linked immunosorbent assays (ELISA)
The production of human IL-6 and IL-10 was measured using ELISA MAX Standard Set (BioLegend). The production of murine CCL2, IL-4, IL-6, IFN-γ, IL-17, TNF-α, and granulocyte–macrophage colony-stimulating factor was measured using ELISA MAX Standard Set kits (BioLegend). Murine IL-2, IL-10, IL-12, CCL3, CCL5 and transforming growth factor-beta production were measured using DuoSet ELISA kits (R&D Systems). Absorbance was determined at 450 nm with subtraction of a 540 nm reference wavelength on iMark™ microplate reader (Bio-Rad laboratories Inc.).
Flow cytometry of human and murine samples
Human PBMC were stained for CD19 (HIB19; BioLegend), CD14 (M5E2; BD Bioscience) and MHC class II (G46-6; BD Bioscience). Composition of murine immune cells was analysed using the following antibodies: CD3 (145-2C11; BioLegend), CD19 (6D5; BioLegend), CD20 (SA275A11; BioLegend), CD11b (M1/70; BioLegend), CD11c (N418; BioLegend), CD45 (30-F11; BioLegend), Ly6C (HK1.4; BioLegend) and Ly6G (1A8: BioLegend). B cell maturation was analysed using the following antibodies: CD19 (6D5; BioLegend), CD21 (7G6; BD Bioscience), CD23 (B3B4; BD Bioscience), CD93 (AA4.1; BioLegend), CD45R/B220 (RA3-6B2; BioLegend), IgD (11-26c.2a; BioLegend) and IgM (AF6-78; BD Bioscience). Monocyte, macrophage and microglia activation, differentiation and molecules involved in antigen presentation were determined using: CD40 (3/23; BD Bioscience), CD68 (FA-11; BioLegend), CD69 (H1.2F3; BioLegend), CD80 (16-10A1; BioLegend), CD86 (GL-1; BioLegend), MHCII (AF6-120.1; BioLegend) and PD-L1 (MIH5; eBioscience). Fc receptors were blocked using monoclonal antibody specific for murine or human CD16/ CD32 (Murine TruStain FcX; Human TruStain FcX; BioLegend), respectively. Dead cells were stained with the Zombie Fixable Viability™ Kit (BioLegend). Samples were acquired on a BD LSR Fortessa (BD Bioscience). All data evaluation was performed using FlowJo software (FlowJo LLC, Ashland, USA).
Cell viability
The WST-1 cell assay (Roche) was used to determine cell viability. After culture, cells received fresh medium containing WST-1 reagent and were incubated at 37° C, 5% CO2 for three hours. Absorbance was determined at 450 nm with subtraction of a 655 nm reference wavelength on iMark™ microplate reader (Bio-Rad laboratories Inc.).
Statistical analysis
Statistics were calculated using GraphPad Prism 6. For the analysis of ex vivo experiments, Gauss distribution was tested via Shapiro–Wilk normality test if n > 6; for experiments with n ≤ 6, a non-Gauss distribution was assumed. For the analysis of in vitro experiments, Gauss distribution was assumed. The respective statistical comparisons used are indicated in the figure legends.
Discussion
We started our investigation on a possible regulatory role of B cells in MS by the simple observation that anti-CD20-mediated depletion of B cells, a highly effective therapy to prevent acute relapses, is associated with an enhanced pro-inflammatory activity of blood monocytes [
20]. Of note, this effect occurred regularly and frequently in individual patients undergoing anti-CD20 treatment. In parallel, we assessed the ability of B cells from these patients to produce regulatory factors, such as anti-inflammatory IL-10. We observed that various CD20-positive B cell subsets can produce anti-inflammatory cytokines and, importantly, the respective ability to do so did not differ from healthy individuals. These findings generated the hypothesis that in patients with MS B cells can control the activity of peripheral and CNS myeloid cells and that this regulatory axis is extinguished by anti-CD20.
In an approach to assess the functional impact of this observation, we determined that the presence of B cells or the supernatant from activated B cells indeed limits the ability of macrophages to be activated in a pro-inflammatory manner. Functionally blocking IL-10 in this system identified this B cell-secreted cytokine as key factor. Performing the identical experiments with microglia generated similar results, highlighting that B cells broadly shape cells of myeloid origin and that B cell regulation may similarly be of relevance within the chronically inflamed CNS. Based on these results, we generated an in vivo setting to study B cell regulation. In the absence of pathogenic B cell function, preventative depletion of B cells via anti-CD20 substantially exacerbated subsequent experimental MS. This deterioration was associated with an unleased activity of macrophages as well as microglia within the CNS. Of note, this enhancement of CNS myeloid cell function similarly occurred when anti-CD20 was initiated in established EAE. Adoptive transfer of B cells back into depleted mice reversed both the clinical deterioration and regulation of CNS macrophages and microglia, while B cells incapable of producing IL-10 failed to do so. In summary, these findings highlight that B cell regulation persists in chronic CNS inflammation and that abrogation of this regulatory axis propagates CNS autoimmune disease via an unleased activity of CNS myeloid cells.
These findings have several implications; in line with other studies [
14,
22], we identified that the identical B cell population can produce pro- as well as anti-inflammatory factors, strictly depending on the stimulus, the respective cellular milieu and largely independent of the B cell differentiation status. This may indicate that the earlier concept of a cellular dichotomy (regulatory vs. pathogenic effector B cells) [
8] may need to be revisited, and that instead, a gradual description of regulatory versus pathogenic B cell properties might be more accurate [
36].
This revised concept generates new perspectives on the role of B cells in various inflammatory conditions including MS. Thus far, the sole presence of immune cells including B cells within the CNS is associated with pathogenic function. In this regard, a better characterization of CNS B cells and plasma cells, including their spatial relation with CNS-located myeloid cells, is required and possibly on the horizon due to novel powerful microscopy and transcriptomic tools [
9].
One central finding of our study is that B cells control the activity of cells of myeloid origin and that pan B cell depletion via anti-CD20 extinguishes this regulatory axis. Hereby, our findings critically complement our knowledge on the immunological consequences of widely used anti-CD20 antibodies. Clearly, a respective clinical impact of this observation may primarily depend on the underlying condition. In MS, however, activated CNS-infiltrated macrophages and microglia are assumed to be key players driving chronic progression within the CNS. In our experimental study, anti-CD20-mediated depletion of B cells activated macrophages and microglia within the CNS, which exacerbated EAE in a model in which B cells are not involved pathogenically. In conjunction, these findings may suggest that while anti-CD20 most effectively prevents de novo CNS immune cell infiltration, lesion formation and thereby clinical MS relapses, it may not be a suitable treatment for the core process of progression independent of focal activity.
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