Journal of Neuroimmunology 270 (2014) 37–44

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New regulatory CD19+CD25+ B-cell subset in clinically isolated syndrome and multiple sclerosis relapse. Changes after glucocorticoids Clara de Andrés a,⁎, Marta Tejera-Alhambra b, Bárbara Alonso b, Lara Valor b, Roseta Teijeiro b, Rocío Ramos-Medina b, Dolores Mateos a, Florence Faure c, Silvia Sánchez-Ramón b,d a

Department of Neurology, Hospital General Universitario Gregorio Marañón, c/Doctor Esquerdo 46, 28007 Madrid, Spain Department of Immunology, Hospital General Universitario Gregorio Marañón, c/Doctor Esquerdo 46, 28007 Madrid, Spain INSERM U932, Institut Curie, Paris, France d Department of Clinical Immunology, Hospital Clínico San Carlos, Madrid, Spain b c

a r t i c l e

i n f o

Article history: Received 27 March 2013 Received in revised form 2 February 2014 Accepted 3 February 2014 Keywords: Multiple sclerosis, cerebrospinal fluid Regulatory B-lymphocytes Perforin expression, methylprednisolone

a b s t r a c t In multiple sclerosis (MS), the immune damage to the central nervous system results from the net balance between self-reactive and immunoregulatory cells, among other factors. We identified novel perforinexpressing regulatory B-cells (BReg) in patients with clinically isolated syndrome, significantly enriched within the cerebrospinal fluid when compared to peripheral blood, of memory B cell phenotype (CD19+CD25+, CD19+CD25+FoxP3+ and CD19+FoxP3+, p = 0.007, p = 0.06 and p = 0.03, respectively). These BReg subsets were also higher in relapsing–remitting MS during relapse symptoms than in non-clinically active MS patients. Suppressive effects by CD19+CD25+hi BReg on CD4+ T cell proliferation seem to be mediated at least in part by perforin/granzyme pathway. To our knowledge, this is the first report that shows cytolytic perforin/granzyme granule storage in B cells; the interesting point is its involvement on BReg cell immunosuppressive mechanisms, similarly to that in TReg cells. Our data may extend the understanding of pathophysiological processes in MS immunoregulation. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS), in which a heterogeneous infiltration of immune cells and soluble mediators within perivascular spaces, CNS parenchyma and cerebrospinal fluid (CSF) plays a relevant role in the pathophysiology (Lucchinetti et al., 2004; Hu and Lucchinetti, 2009). Increasing evidence fosters a key role of B cells in MS pathophysiology, reinforced by the presence of follicle-like structures in the meninges described in some MS patients (Serafini et al., 2004). B-cells have a complex role in the immune response, both as antigen presenting cells priming T-cell differentiation and as effector cells, producing antibodies and thus autoantibodies, cytokines, adhesion molecules and chemokines that induce leukocyte infiltration and proangiogenesis; and other molecules involved in tissue repair (Porakishvili et al., 2001; Silverman and Carson, 2003; Anderton and Fillatreau, 2008; Kessel et al., 2012).

⁎ Corresponding author at: Department of Neurology, Hospital General Universitario Gregorio Marañón, Calle Doctor Esquerdo 46, E-28007 Madrid, Spain. Tel.: +34 91 586 8339; fax: +34 91 586 8018. E-mail address: [email protected] (C. de Andrés).

http://dx.doi.org/10.1016/j.jneuroim.2014.02.003 0165-5728/© 2014 Elsevier B.V. All rights reserved.

The subset of CD20+CD25+ B cells exhibits immunoregulatory properties (Agematsu et al., 2000). In healthy subjects, these and other authors described that the majority of CD27+ memory B-cells coexpress the alpha chain of IL-2R (CD25+) compared to CD27+ expression on around the 10% of naïve B-cells and plasmablasts, and that CD25+ B-cells display better antigen-presenting cell capacity and secrete significantly higher levels of the immunosuppressive cytokine IL-10 than CD25− B cells (Agematsu et al., 2000; Brisslert et al., 2006; Amu et al., 2007; Kessel et al., 2012 Jul; 66(1):77–86). In addition, this subset expresses more surface IgA and IgG levels than its CD25− counterpart, but lacks the ability to secrete them. Recently, diverse BReg subsets have been characterized, such as IL-10- and TGF-βproducing BReg with CD19+CD5+IgM+ hiIgD+CD1d+ hi phenotype in allergic and autoimmune diseases (Lund and Randall, 2010; Villar et al., 2010; Noh and Lee, 2011). Cytokine-mediated suppressive effects seem to operate in this subset, while cell-mediated mechanisms have not been described. Among the latter, the perforin/ granzyme-dependent lytic pathway has been shown to mediate suppression by regulatory TReg (Valor et al., 2011). In order to ascertain whether perforin/granzyme may play a role in BReg regulatory function, we studied the expression of this molecule by BReg (Tejera-Alhambra et al., 2012). The first clinical episode suggestive of MS was defined as clinically isolated syndrome (CIS), while MS relapse usually occurs in already

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diagnosed relapsing–remitting MS form (RRMS). An active recruitment of plasmacytoid dendritic cells (pDCs) and TReg into the CSF during relapse (De Andrés et al., 2000; Fritzsching et al., 2011; SánchezRamón et al., 2004), and their accumulation in white matter lesions and leptomeninges of MS patients have been demonstrated, suggesting that different immune cells interplay in the complex MS pathophysiology. Only limited data, however, are available regarding the distribution of B-cell subsets in blood and CSF, and in the setting of MS relapse, as well as their putative interactions with other immune cells, or on the effect of glucocorticoid (GC) treatment for MS relapse on the B cell compartment. GC remains the gold-standard therapy for acute relapses of MS and other numerous autoimmune diseases. Their mechanisms of action, however, have not been completely elucidated yet. GCs act through different genomic and non-genomic mechanisms that are mediated by the binding to cytosolic hormone as well as to cell membrane receptors, or by directly interacting with enzymes and other cell proteins. The immunosuppressive and anti-inflammatory effects of GCs are mainly mediated through induction or repression of gene transcription (Stahn and Buttgereit, 2008). GCs exert dual inhibitory and stimulatory effects on a number of components of the immune response, via secretion of cytokines, chemokines and adhesion molecules; interfering with the BBB disruption; and favoring extracellular matrix remodeling (Tischner and Reichardt, 2007). We and others have previously described in vivo and in vitro GC effects on DCs and TReg (Navarro et al., 2006) and on CD8+ T cell subsets (Aristimuño et al., 2008), and on total CD19+ B cells (Krystyna et al., 2009). Here we performed two separate studies: a cross-sectional study of CIS patients during relapse to simultaneously evaluate PB and CSF distribution of distinct BReg subsets; and a prospective observational study in recurrent–remitting MS (RRMS) patients during MS relapse to assess short-term effects of GC therapy on B-cell subsets. 2. Materials and methods 2.1. Patients Thirty-seven RRMS patients with clinically-definite diagnosis (21 during MS relapse (MSR-group) and 16 in clinical remission and untreated (MS controls, MSC-group)) and 17 patients with CIS symptoms (CIS-group) that were consecutively referred to the Neurology Dept. at the Hospital General Universitario Gregorio Marañón were studied. CIS is a term that describes a first clinical episode with features suggestive of MS (Miller et al., 2012). We performed two different studies: In the CIS-group, PB and CSF cell subsets were studied in parallel to compare immunoregulatory B cell distribution; and a second study in MSR-group to analyze the variation of immunoregulatory B cells before and after short-term IV-MP therapy for MS relapse (Post-IV-MP-group). An additional control group consisted of 26 age-adjusted healthy volunteers (HC-group, 18 female and 8 male). Clinical disease severity was scored by the Kurtzke's Expanded Disability Status Scale (EDSS) (Kurtzke, 1983). All MS patients presented with white matter lesions on brain and/or spinal cord magnetic resonance imaging (MRI) and laboratory data (IgG index, and/or oligoclonal IgG bands consistent with McDonald MS criteria (Polman et al., 2011)). MS relapse was defined as the occurrence of new neurological symptoms or worsening of pre-existing neurological symptoms lasting more than 24 h after a period of 30 days of improvement or stability in the absence of infection or fever. The MSR-group was evaluated at two different time-points, during relapse and after 3 to 5 days of iv methylprednisolone therapy (IV-MP, 1 g/day). All blood and CSF samples were drawn within 4 weeks from the onset of MS symptoms (median, 10.0 days; range, 2 to 23 days). Patients with kidney, liver, endocrine or infectious disorders were excluded by history, physical examination, and laboratory evaluations. None of the MS patients had received any immunosuppressive

treatment for at least 2 months prior to this study time-point. Informed consent was obtained from all participants. The Local Ethics Committee approved this study, and all patients and healthy donors provided informed consent. 2.2. Blood sampling and CSF preparation of samples and isolation of peripheral blood mononuclear cells Peripheral blood (PB) samples were collected by venipuncture into sodium heparin and EDTA VACUTAINER® tubes (Becton Dickinson, New Jersey, USA) from all groups of patients and HC. In the CIS-group, the CSF samples were simultaneously analyzed. PB and CSF samples were processed within 2 h of collection from all groups of patients. Patients with active MS relapse (MSR-group) were studied before the initiation of IV-MP therapy and after 3–5 days of IV-MP (1 g/day). Briefly, whole peripheral blood of CSF samples (100 μl) was labeled by direct staining with monoclonal antibodies (mAbs) (CD19−PerCPCy5/CD25−APC, Becton Dickinson, Mountain View, CA, USA), incubated for 30 min at 4 °C, washed twice and fixed in Cell-fix (Becton Dickinson, Stockholm, Sweden). For intracellular staining with perforin-PE (BD, Pharmingen) anti-FoxP3-Alexa Fluor® 488 (BD, Pharmingen) and granzyme-PE (BD, Pharmingen), cells were first labeled with anti-CD25-APC and CD19-PerCpCy5. Cells were then fixed, permeabilized, and stained with anti-perforin/granzyme-PE and FoxP3-Alexa Fluor®488 following manufacturer's instructions. Isotype control mAbs were used to determine background fluorescence levels. Acquisition and analysis were performed in a FACScalibur flow-cytometer (Becton Dickinson), using a CellQuest Software. A gate was drawn to include the lymphocytes in a dot plot of Forward Scatter (FSC) versus Side Scatter (SSC), and a total of 50,000 events were collected (R1). For analysis of the regulatory CD19+ lymphocytes (BReg), a second gate on a side scatter/ CD19 −PerCPCy5 dot plot was set up to collect total B cells (R2). B Reg were defined as CD19 + CD25 + FoxP3 + , CD19 + FoxP3 + and CD19+CD25+bright. The content of intracellular perforin was measured in CD19+CD25+FoxP3+ cells and CD19+FoxP3+ cells (Fig. 1). 2.3. CD19+ regulatory B cell purification and co-cultures with T cells PBMC were isolated from buffy coats of healthy donors within 40 min with Lymphoprep (Nycomed, Norway) according to manufacturer's instructions. CD19+ B and CD4+ T cells were purified with the cytometer MoFlo XDP Cell Sorter (Beckman Coulter, Inc.) by the enrichment method. In a second step, CD19+CD25hi Breg cells and CD19+CD25− were sorted. The purity obtained was N 95%. CD4+ T cells (10 × 104) were cultured alone, co-cultured with CD19+CD25hi B cells at ratio 1:2 (50 × 103), or co-cultured with CD19+CD25− B cells at the same ratio (1:2). CD19+CD25− B cells (10 × 104 cells) were cultured alone to control spontaneous proliferation. All wells were seeded in 96-well round-bottom plates in RPMI 1640 medium supplemented with 10% FCS, glutamine, penicillin, streptomycin, amphotericin and HEPES buffer for 4 days. Once cultured, T cells were stimulated with anti-CD3 mAb (1 μg/ml) and anti-CD28 mAb (1 μg/ml) both from Becton Dickinson (New Jersey, USA); B cells were stimulated with CD40L (0.5 μg/ml), enhancer (1 μg/ml), and IL-4 (5 μg/ml). Cell proliferation was measured by 3[H]Thymidine (3[H]T) (1 μCi/well) by Matrix 96 Direct Beta Counter Packard. The counted radioactivity of proliferating cells was expressed as counts per min (cpm). 2.4. Degranulation assays Breg and NK-cell degranulation functions and perforin/granzyme expression were tested in a single-cell assay using CD107a+b (LAMP) mobilization, as previously described (Betts et al., 2003). In brief, PBMC from an MS patient and a healthy control were cultured in

C. de Andrés et al. / Journal of Neuroimmunology 270 (2014) 37–44

39

Fig. 1. Proportions of CD19+ B and BReg lymphocytes in the cerebrospinal fluid (CSF) and in peripheral blood (PB) during the first clinically isolated syndrome (CIS). Gating strategy used to define BReg lymphocyte subset detection (CD19+CD25+FoxP3+) and its expression of perforin. CSF and peripheral blood mononuclear cells were selected in the R1 gate (acquiring up to 50,000 events in PB) excluding debris and polynuclear cells. CD19 positive cells were selected in the R2 gate and then, BReg were defined as CD19+CD25+FoxP3+ (R3). Finally, expression of perforin positive cells in R3 gate. The upper panel shows that proportions of BReg, defined as CD19+FoxP3+CD25+, were higher in CSF than in PB. Both sides of Breg the proportions of regulatory CD4+CD25+FoxP3+ TReg of the same patient are shown. This lower panel shows a representative example of the higher expression of perforin on CD19+FoxP3+CD25+ cells in the CSF than in the PB. In the histogram, the MFI is compared to isotype control (in red).

complete RPMI-1640 medium (Biochrom AG, Berlin, Germany) at 106 cells/ml without any antigenic stimuli (negative control) or with the following stimuli: PMA (2 μg/ml)/ionomycin (1 μM; SigmaAldrich; St. Louis, Missouri); and IL-4 (5 μg/ml) plus CD40L (0.5 μg/ml) and enhancer (1 μg/ml). After 1 h of incubation at 37 °C and 5% CO2, cells were collected and stained with anti-CD3, anti-CD25, anti-CD16, anti-CD56, anti-CD19, anti-perforin, anti-granzyme and anti-CD107a + b (BD Biosciences, San José, California). A total of 100,000 events were analyzed by flow cytometry in the lymphocyte gate. Percentages of BReg and NK cells were evaluated simultaneously for CD107a + b, granzyme and perforin expression using the abovedescribed cell gating strategy. For the intracellular perforin staining PBMC were fixed/permeabilized (Fix/Perm, BD Biosciences), and stained with Perforin and Granzyme. Cytometry data were analyzed with FlowJo software (Tree Star, Ashland, OR).

3. Results 3.1. Demographic and clinical characteristics of MS patients The demographic and clinical characteristics of the 54 MS and CIS patients at baseline are shown in Table 1. The patients' selection and time of testing for immune populations were at first neurological exam at the hospital and before initiating therapy. The number of days after clinical onset of symptoms ranged between 2 and 23 days (mean, 10 days). Patients with clinically active MS relapse

(MSR-group) were studied before the initiation of IV-MP therapy and after 3–5 days of IV-MP (1 g/day). 3.2. CSF and peripheral blood proportions of regulatory CD19+ lymphocytes in CIS patients during relapse In a group of 17 patients during the symptoms of CIS, we investigated proportions of different subsets of BReg (Table 2) as well as regulatory CD4+ T-cell subsets in parallel in CSF and PB. Proportions of TReg were higher in CSF than their PB counterparts (p = 0.05 for TReg), as previously described by other authors and by us (Feger et al., 2007; Table 1 Epidemiological and clinical characteristics of the groups of patients studied, namely clinically isolated syndromes (CIS) (n = 17) for the cross-sectional study; MS patients during relapse, MSR-group (n = 21) and after short-term IV-MP therapy; and control MS patients with clinical remission, MSC-group (n = 16).

No. of patients Age, median years (range) Disease duration, median years (range) Number of previous relapses, median (range) EDSS, median (range) Gender, female/male ratio

CIS-group

MSR-group

MSC-group

17 38 (21–51) 0

21 34.4 (21–45) 6.2 (1–16)

16 33.3 (19–45) 5.3 (1–15)

0

4.2 (1–9)

3.2 (1–6)

1.5 (1–3) 13/4

3.07 (1.5–5.5) 17/4

0.46 (0–3.5) 10/6

EDSS: Expanded Disability Status Scale (Kurtzke, 1983).

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Table 2 Proportions of the different BReg subsets in CSF and PB of patients during CIS symptoms. N = 17

CD19+

CD19+CD25+

CD19+CD25+FoxP3+

CD19+FoxP3+

CSF PB p

4.13 ± 5.29 9.58 ± 2.74 0.001

30.75 ± 20.86 15.91 ± 11.31 0.007

3.20 ± 6.21 0.17 ± 0.12 0.06

11.87 ± 20.64 0.34 ± 0.21 0.03

Data are given as mean ± SD.

Fritzsching et al., 2011; Tejera-Alhambra et al., 2012). B lymphocytes co-expressing the activation molecule CD25 were more abundant in the CSF than in PB (30.75 ± 20.86 vs 15.91 ± 11.31, p = 0.007) (Table 2 and Fig. 2). Similarly, BReg co-expressing FoxP3+ were significantly higher in CSF than in PB (11.87 ± 20.64 vs 0.34 ± 0.21, p = 0.03) and the proportions of CD19+CD25hi+FoxP3+ BReg were more abundant in CSF than in PB (3.20 ± 6.21 vs 0.17 ± 0.12, p = 0.06) (Table 2). Interestingly, perforin expression on CD19+CD25hi+FoxP3+ BReg and CD19+CD25+ memory B-cells within the CSF was significantly higher than that in PB (p = 0.003 and p = 0.005, respectively) (Table 2). The majority of CD19+FoxP3+ lymphocytes present in the CSF expressed perforin (median, (IQR) of 100.00% (36.61) vs 11.76% (32.82), p = 0.01) with respect to this lymphocyte subset in PB. In addition, we found higher intracellular granzyme expression and surface expression of CD107a + b (LAMP-1 and -2) molecules by ex vivo CSF BReg that indicate actual cell degranulation by these cells (data not shown). In summary, we report that during CIS symptoms, patients have increased percentages of both memory B cells and BReg in CSF compared to PB and that BReg preferentially express and excrete perforin/granzyme within the CNS compartment. 3.3. Distribution of peripheral B cell subsets (CD19+, CD19+CD25+FoxP3+, CD19 +CD25+ and CD19 + FoxP3 +) in MS patients during relapse (MSR-group) and after iv methylprednisolone treatment We observed that the proportions of circulating regulatory CD19+CD25+FoxP3+ and CD19+FoxP3+ BReg in the MSR were higher than in the MSC-group (p = 0.05 and p = 0.01, respectively) (Table 3). After IV-MP for relapse in MSR patients, we observed an increment on the proportion of total circulating CD19+ B-cells (p b 0.0001) (Table 3). In contrast, a decrease in the proportions of regulatory

CD19 +CD25 +FoxP + and CD19 +FoxP3 + after IV-MP (p = 0.006 and p = 0.004, respectively) was observed (Table 3). Of note, the subset of memory CD19+CD25+ was higher in MSC with respect to HC and to MSR (p = 0.01 and p = 0.0001, respectively). In the MSR-group, a strong positive correlation was observed between the proportions of CD19+CD25+FoxP+ BReg with CD4+CD25+FoxP3+ TReg at MS relapse (r = 0.773, p b 0.0001). Also, perforin expression on BReg directly correlated with that on TReg, r = 0.775, p b 0.0001. 3.4. BReg suppress proliferation of co-cultured CD4+ T cells Blood CD19+CD25+ B cells in the MSR-group disclosed a distinct memory B phenotype than that of their CD25− counterpart, being approximately 60% of the memory B CD27+IgD− phenotype versus predominantly naïve the CD25− subset (data not shown). Compatible with that previously described (Kessel et al., 2012), BReg inhibited the proliferation of co-cultured autologous TCR-stimulated CD4+ T cells. Our results in three independent experiments in healthy controls showed a 71.60% suppression of proliferation of CD4+ T cells in the presence of BReg than that with CD4+CD25− B cells at the same ratio (Fig. 3). 3.5. Degranulation assay To further characterize the expression of perforin by Breg, we performed a degranulation assay on PBLs of a healthy control and an MS patient to compare with cytotoxic NK cells. PBLs were stimulated in parallel with PMA- and CD40 plus CD40L for 1 h. Non-stimulated NK cells expressed high levels of CD107a+b (LAMP-1 and -2) whereas stimulation with PMA/IM greatly enhanced perforin and granzyme expression. In contrast, non-stimulated BReg did not show CD107a+ b expression that was highly upregulated after stimulation, with mild increase of both granzyme and perforin, suggesting perforin loss due to degranulation (Fig. 4). No differences were observed between the MS patient and the healthy control. 4. Discussion Cumulative evidence supports a relevant role of B cells and their products in the pathogenesis of MS. Indeed, oligoclonally expanded B cells were found in brain lesions and the CSF of MS patients

Table 3 Short-term prospective study comparing the percentages of peripheral B cell subsets (CD19+, CD19+CD25+FoxP3+, CD19+CD25+ and CD19+FoxP3+) and their expression of perforin in healthy controls (HC-group), MS untreated non-relapse (MSC-group), and MS during relapse (MSR-group) and after IV-MP treatment (Post-IV-MP-group). Statistically significant effects are marked in bold. B cell subsets

HC-group

MSC-group

pa (HC vs MSC)

MSR-group

MS Post-IV-MP-group

pb (MSR vs post-IV-MP)

pa (MSR vs HC)

pa (MSR vs MSC)

CD19+

11.52 ± 4.00 10.58 (9.78–13.80) 0.28 ± 0.19 0.25 (0.13–0.41) 17.97 ± 15.40 13.64 (7.14–25.33) 25.40 ± 15.92 22.14 (14.14–32.64) 19.89 ± 14.48 15.73 (9.72–30.28) 2.23 ± 5.30 0.70 (0.29–1.67) 85.71 ± 245.30 20.16 (15.76–47.62) 1.40 ± 2.55 0.62 (0.40–1.10) 76.81 ± 183.01 24.68 (16.18–53.09)

8.50 ± 3.24 7.73 (5.38–11.83) 0.40 ± 0.27 0.32 (0.17–0.61) 31.61 ± 27.32 23.23 (8.66–55.56) 89.62 ± 152.40 38.15 (33.49–55.42) 31.32 ± 15.19 35.38 (17.26–43.32) 1.37 ± 1.24 0.76 (0.43–2.08) 114.52 ± 191.51 42.98 (30.72–91.31) 0.90 ± 0.61 0.79 (0.38–1.22) 210.91 ± 368.12 39.05 (30.41–327.13)

0.05

10.86 ± 5.12 9.41 (7.96–13.69) 0.88 ± 3.07 0.18 (0.70–0.35) 30.21 ± 25.96 19.35 (6.79–54.94) 29.82 ± 20.88 25.90 (21.70–36.81) 11.94 ± 11.54 7.84 (2.77–18.97) 4.48 ± 12.88 0.88 (0.38–2.54) 29.82 ± 20.88 25.90 (21.70–36.81) 2.82 ± 10.81 0.45 (0.16–0.61) 36.32 ± 24.66 26.25 (22.54–47.88)

17.34 ± 6.00 16.28 (12.79–21.64) 0.10 ± 0.12 0.04 (0.01–0.15) 16.20 ± 26.42 0.00 (0.00–22.39) 37.27 ± 44.62 20.69 (10.09–54.50) 15.22 ± 12.76 13.15 (5.59–19.64) 0.47 ± 0.53 0.38 (0.00–0.84) 36.56 ± 36.87 24.47 (15.12–48.18) 0.26 ± 0.44 0.11 (0.05–0.27) 231.79 ± 625.63 52.68 (20.90–154.50)

b0.0001

NS

NS

0.006

NS

0.05

0.08

NS

NS

NS

NS

0.004

NS

0.05

0.0001

NS

NS

NS

NS

NS

0.007

0.004

0.05

0.01

0.07

NS

0.02

CD19+CD25+FoxP3+ %Perforin MFI perforin CD19+CD25+ %Perforin MFI perforin CD19+FoxP3+ MFI perforin

NS NS 0.001 0.01 NS 0.05 NS 0.01

Data are presented as mean ±SEM, median (IQR). a Sig. (2-tailed) between MSR-group and HC- and MSC-groups and between HC-group and MSC-group by Mann–Whitney U Test. b Sig. (2-tailed) between MSR-group and Post-IV-MP-group by Wilcoxon Signed Rank Test.

C. de Andrés et al. / Journal of Neuroimmunology 270 (2014) 37–44

Fig. 2. Differences in the blood B and BReg cell subset size in the MSR-group at relapse and after short-term IV-MP therapy (Post-IV-MP-group), compared with healthy controls (HC-group) and MS patients with clinical remission (MSC-group). Box plot showing the frequencies of B cells (%CD19+) and of activated B cells (%CD19+CD25+) in all groups studied. Each box plot represents the median (thick band) and the 25th and 75th centiles. The error bars represent the smallest and largest values that are not outliers. The symbols represent outliers.

(Qin et al., 1998). These CNS-resident B cells have been linked to the production of intrathecal antibodies of restricted specificities, detectable as oligoclonal bands (OCB). Moreover, persistent intrathecal

Fig. 3. Suppression of CD4+ T cell proliferation after coculture with CD19+CD25+ B cells. The proliferation of TCR-stimulated CD4+ T cells was suppressed in the presence of BReg (CD19+CD25+ cells) in 71.60% in 3 independent experiments.

41

IgG and IgM OCB presence in the CSF has been reported as a worse prognostic marker (Villar et al., 2010). B-cell follicles have been described in the meninges of patients with secondary progressive MS and ectopic structures resembling germinal centers have been found in the brains of MS patients (Serafini et al., 2004), histological findings demonstrating Ig and complement deposit in inflammatory brain lesions (Lassmann et al., 2007). Indeed, detection by double immunofluorescence of IgM and IgG colocalized with C3b complement on axons and oligodendrocytes in demyelinated areas and on degenerating myelin sheaths in necropsies from a series of MS patients (Sadaba et al., 2012); and also IgM-Ab recognized neuronal surface antigens, and the levels of neuronal-bound IgM-Ab correlated with brain atrophy on MRI in MS patients (Beltran et al., 2012). The phenotype of B-cells at both sides of the blood–brain barrier (BBB) has been recently investigated in MS (Cepok et al., 2005; von Budingen et al., 2011). These authors observed that most B cells within the CSF were of the CD19+CD27+CD138− memory phenotype, but some cells were CD19+CD138+, indicating their differentiation into plasma cells (plasmablasts). Brisslert et al. (2006) were the first who suggested that PB CD19+CD25+ B cells coexpress CD27+ memory molecule and have a regulatory role. CD19 +CD25 + B cells were characterized by expressing high levels of surface Ig compared with CD19 +CD25 − B cells, but they lacked the ability to secrete them, and also expressed high levels of costimulatory molecules. Moreover, they were shown to secrete high levels of inhibitory cytokine IL-10 compared with CD25 − B-cells and to present antigen more efficiently to allogeneic CD4+ T-cells in mixed lymphocyte reaction compared with CD25− B cells, a function that was attributed to the high expression level of CD25 (Amu et al., 2007). Recently, Eriksson et al. (2010), observed that in patients with Wegener's granulomatosis during remission, the proportion of circulating CD25+ B cells was increased compared to controls and to patients with active vasculitis, and Knippenberg et al. (2011) described that during MS relapse, IL-10+ BReg and naïve/memory B-cells were reduced in PB. In accordance with other autoimmune diseases during remission (Eriksson et al., 2010), we found that non-active RRMS (MSC) patients disclosed significantly increased proportions of circulating memory CD19+CD25+ B cells with respect to healthy controls and to MSRgroup. We can speculate that MSR-group patients could show lower levels of CD19+CD25+ B cells due to their immigration into the CSF in response the inflammatory microenvironment developing within the CNS, as we have observed in the group of CIS patients, where a differential recruitment of CSF CD19+CD25+ B cells was noted. With regard to their function, CD25+ B cells seem to work as immunomodulatory B cells by secreting the immunosuppressive cytokine IL-10 (Brisslert et al., 2006; Kessel et al., 2012). Furthermore, FoxP3 and CTLA-4 expression in TReg cells was enhanced by non-stimulated direct contact. The regulatory function of BReg cells on TReg cells was mainly dependent on a direct contact between BReg and TReg cells, although it was also TGF-β-, but not IL-10-dependent. In accordance with previous authors, we confirmed regulatory suppressive function of the CD19+CD25+hi BReg on activated CD4+ T-cells as a significant reduction of their proliferation that may be due at least in part to the expression and degranulation of the perforin/granzyme molecule. BReg recruitment with the CNS could thus also contribute to MS pathophysiology. To our knowledge, this is the first report that shows cytolytic perforin/granzyme granule storage in B cells; the interesting point is its involvement on B Reg cell immunosuppressive mechanisms, similarly to that in TReg cells. The exact contribution of different subsets of regulatory B cells including the CD19+CD25+FoxP3+ to MS pathogenesis has not been thoroughly studied. There was limited knowledge on the distribution and significance of distinct B-cell subsets within PB and CSF during and beyond phases of disease activity, or to their changes during the MS treatment, such as the glucocorticoids for MS relapse. The best evidence for inflammation during relapses comes from MRI that

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Fig. 4. Degranulation assay. Expression of granzyme and perforin and CD107a+b (LAMP-1 and -2) in BReg and NK cells was measured by analysis of these markers by flow cytometry. PBMC of a healthy donor (HC) and an MS patient were stimulated with PMA/IM to analyze intracytoplasmic perforin and granzyme, and then we compared these results with CD107a+b (LAMP-1 and -2) expression by Breg and NK cells ex vivo (red line) and after stimulation (blue line). CD107a+b was highly expressed by NK cells, with high expression of perforin and granzyme with respect to BReg cells after stimulation. In BReg, CD107a+b positive cells 1 h post-stimulation possess lower perforin/granzyme with respect to nonstimulated cells, suggesting perforin loss due to degranulation. The percentages given correspond to PMA/IM stimulated cells.

demonstrates the association of clinical relapses with gadolinium enhancement that is due to disruption of the BBB. During relapses, the inflammatory microenvironment developing inside the CNS is involved in leukocyte recruitment and could be linked to intrathecal CXCL13concentrations (Sellebjerg et al., 2009). These authors observed that CNS-recruitment of B cells was linked to upregulation of CXCL12 and CXCL13 chemokines in the CSF and in active MS plaques, and to the expression induction of different integrins (α4 and β1). In particular, memory B-cells can express high concentrations of the adhesion molecule very-late antigen (VLA)-4 that binds to vascular-cell adhesion molecule-1 on the endothelial cells (Niino et al., 2006; Lee-Chang et al., 2011). We showed here that during CIS symptoms, the CD19+CD25+ memory and CD19+CD25FoxP3+ and CD19+FoxP3+ BReg were enriched in CSF compartment with respect to PB. At MS relapse (MSR-group), we found that CD19+CD25+FoxP3+ and CD19+FoxP3+ BReg were significantly decreased in PB with respect to MSC, probably due to immigration into the CNS as an immunoregulatory mechanism to control the MS activity. Compatible with previous studies (Corcione et al., 2004; Lund and Randall, 2010; Kessel et al., 2012), we confirmed that BReg can modulate the immune response through suppression of activated CD4+ T cells, and thus enhance the effects of other regulatory subsets, such as regulatory CD4+ T cells with whom they strongly correlate. We also found that CSF of patients with CIS and other inflammatory neurological diseases was enriched in class-switched CD27+IgD−IgM− BReg, suggesting that they were probably originated from the peripheral compartment. Recently (Lisak et al., 2012), an in vitro study has demonstrated that B cells from patients with RRMS but not from HC secrete toxic factors to

oligodendroglia, and suggest that, it was possible that such secretory factors were produced by peripheral blood B cells within the CNS which could contribute to demyelination in MS patients. Not previously reported, we found perforin molecules specifically expressed in regulatory B-cell subsets in MS patients, preferentially in CD19+FoxP3+ BReg, molecule involved in cell-contact-mediated suppression and MS pathophysiology. Perforin is a cytolytic pore-forming glycoprotein of 534 aa that supposedly generates in target cell membrane polyprotein pores of 12–18 perforin monomers, allowing the entry of granzyme and results in target cell death by apoptosis (Lichtenheld et al., 1988; Trapani and Smyth, 2002). Perforin molecules are stored within the cytoplasmic lytic granules of two of the main cytotoxic cell populations, CD8+ T cells and NK cells (Chavez-Galan et al., 2009), and in TReg as described by us, with relevant role in MS (TejeraAlhambra et al., 2012) and contribute to T cell homeostasis. A limitation of the study was the lack of killing assay by classic chromium release assay (CRA), due to the limitations of the CRA assay for the requirement of a large amount of cells, which are difficult to obtain considering the clinical characteristics of our patients (MS patients). In MSR, high proportions of circulating regulatory CD19 + CD25 +FoxP3+ and CD19+ FoxP3 + B Reg with respect to MSC were observed, probably as a compensatory peripheral response to the inflammatory circumstances of relapse. After IV-MP treatment for relapse in MSR patients, we observed an increment on the proportion of circulating total CD19+ B-cells, as in other authors (Krystyna et al., 2009), while decreased proportions of CD3+ T lymphocytes (data not shown) as in our previous study (Navarro et al., 2006). Following IV-MP treatment, a reduced amount of soluble VCAM-1 and E-selectin

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in the serum of MS patients has been shown (Elovaara et al., 2000) that could reduce the migration of these cells into CNS, as glucocorticoids inhibit the disruption of the BBB and thereby reduce leukocyte infiltration into the CNS (Paul and Bolton, 1995). We also observed a decrease in the proportion on the regulatory CD19+CD25+FoxP+ and CD19+FoxP+ after IV-MP. Also, the receptor of corticosteroids GITR is constitutively expressed on B-cells and has been implicated in regulating both innate and adaptive immune responses. In conclusion, the main findings of the current study have been the identification of a novel regulatory B-cell subset in small but appreciable quantities in the PB of HC and in MS patients, but greatly enriched within the CSF during clinical manifestations. First, the MS patients in remission (MSC-group) have increased circulating CD19+CD25+ memory B cells as compared to HC, probably due to previous MS antigen interaction, and lower proportion of CD19+FoxP3+ BReg than MS during relapse (MSR-group), probably due to their immigration into CNS. Second, during CIS symptoms, higher proportions of CSF memory B and different BReg subsets than PB were seen. Third, the suppressive function of BReg on proliferation of activated CD4+ T cells may be also mediated by perforin/granzyme pathway. Fourth, in MSR after IV-MP for the relapse treatment, we observed an increment of total circulating CD19+ mature B cells while decreased BReg subsets. The understanding of pathophysiological processes in the regulation of autoimmune disease, and MS in particular and the critical role of BReg in the periphery and within the CNS, are relevant to understanding the pathogenesis of MS and in the design of novel therapeutic strategies. Disclosures The authors declare that they have no competing financial interest. Acknowledgments We would like to thank our patients with MS and the healthy volunteers that generously contributed blood and CSF samples to this study. We wish to thank Haydee Goycochea for excellent nursing care of our patients and Jose Mari Bellón for help in statistical analyses. The authors would like to thank William Morosse for the English language review. This study was supported by grants from the Fundación Alicia Koplowitz and Fundación Salud 2000 through the Instituto de Investigación Gregorio Marañón to SS. References Agematsu, K., Hokibara, S., Nagumo, H., Komiyama, A., 2000. CD27: a memory B-cell marker. Immunol. Today 21, 204–206. Amu, S., Tarkowski, A., Dorner, T., Bokarewa, M., Brisslert, M., 2007. The human immunomodulatory CD25 + B cell population belongs to the memory B cell pool. Scand. J. Immunol. 66, 77–86. Anderton, S.M., Fillatreau, S., 2008. Activated B cells in autoimmune diseases: the case for a regulatory role. Nat. Clin. Pract. Rheumatol. 4, 657–666. Aristimuño, C., Navarro, J., de Andres, C., Martinez-Gines, L., Gimenez-Roldan, S., Fernandez-Cruz, E., Sanchez-Ramon, S., 2008. Expansion of regulatory CD8 + T-lymphocytes and fall of activated CD8 + T-lymphocytes after i.v. methylprednisolone for multiple sclerosis relapse. J. Neuroimmunol. 204, 131–135. Beltran, E., Hernandez, A., Lafuente, E.M., Coret, F., Simo-Castello, M., Bosca, I., Perez-Miralles, F.C., Burgal, M., Casanova, B., 2012. Neuronal antigens recognized by cerebrospinal fluid IgM in multiple sclerosis. J. Neuroimmunol. 247, 63–69. Betts, M.R., Brenchley, J.M., Price, D.A., De Rosa, S.C., Douek, D.C., Roederer, M., Koup, R.A., 2003. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods 281 (1–2), 65–78 (Oct 1). Brisslert, M., Bokarewa, M., Larsson, P., Wing, K., Collins, L.V., Tarkowski, A., 2006. Phenotypic and functional characterization of human CD25+ B cells. Immunology 117, 548–557. Cepok, S., Rosche, B., Grummel, V., Vogel, F., Zhou, D., Sayn, J., Sommer, N., Hartung, H.P., Hemmer, B., 2005. Short-lived plasma blasts are the main B cell effector subset during the course of multiple sclerosis. Brain 128, 1667–1676. Corcione, A., Casazza, S., Ferretti, E., Giunti, D., Zappia, E., Pistorio, A., Gambini, C., Mancardi, G.L., Uccelli, A., Pistoia, V., 2004. Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A. 101, 11064–11069.

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