Interferon-α induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus

Journal of Autoimmunity xxx (2015) 1e11

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Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus Nan-Hua Chang a, Timothy T. Li a, Julie J. Kim b, Carolina Landolt-Marticorena a, Paul R. Fortin c, Dafna D. Gladman a, Murray B. Urowitz a, Joan E. Wither a, * a b c

Arthritis Centre of Excellence, Toronto Western Hospital Research Institute, University of Toronto Faculty of Medicine, Toronto, Canada University of Ottawa, Ottawa, Canada CHU de Qu ebec and Faculty of Medicine, Universit e Laval, Quebec City, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 September 2014 Received in revised form 22 December 2014 Accepted 19 January 2015 Available online xxx

Previous studies suggest that the B cells of patients with Systemic Lupus Erythematosus (SLE) are hyperresponsive to BCR crosslinking; however, it has been unclear whether this is the result of altered B cell signaling or differences in various B cell subpopulations in SLE patients as compared to healthy controls. Here we have developed a novel Phosflow technique that permits examination of cell signaling in distinct B cell subpopulations stratified based upon developmental stage and cell surface IgM levels, which we use to show that the naïve B cells of SLE patients are hyper-responsive to IgM receptor crosslinking, resulting in increased SYK phosphorylation. We further demonstrate that this hyperresponsiveness is most marked in the transitional B cell subset and that it is associated with altered function, resulting in decreased apoptosis and increased proliferation of these cells. Examination of repeated samples from the same patients revealed that the hyper-responsiveness fluctuated over time, suggesting that it may be mediated by pro-inflammatory factors rather than genetic variations between patients. In support of this concept, incubation of healthy control B cells with IFN-a or SLE plasma induced the hyper-responsive phenotype, which was blocked by anti-IFN-a antibody. Furthermore, no obvious correlation was seen between genetic variants that are proposed to alter BCR signaling and the increased SYK phosphorylation. The findings suggest that pro-inflammatory factors, in particular Type I IFNs, modulate B cell function in SLE in a way that could contribute to the breach of tolerance in this condition. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Lupus B cell SYK Hyper-responsive Interferon-alpha

1. Introduction The presence of diverse auto-antibodies (Abs) in Systemic Lupus Erythematosus (SLE) suggests that the mechanisms that prevent activation of self-reactive B cells are defective. Studies in mice indicate that the strength of the B cell receptor (BCR) signal plays an important role in determining the fate of auto-reactive B cells, with genetic manipulations that enhance or impair BCR signaling, promoting lupus-like autoimmunity [1e8]. Recently, several lupus risk variants for genes that encode molecules downstream of the BCR

Abbreviations: Abs, antibodies; SLE, Systemic Lupus Erythematosus; BCR, B cell receptor; IFN, interferon; p-, phospho-. * Corresponding author. 1E420, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada. Tel.: þ1 416 603 5048; fax: þ1 416 603 4348. E-mail address: [email protected] (J.E. Wither).

have been described that could lead to altered B cell signaling in humans [9e14]. While the precise impact of some of these variants on B cell signaling remains to be determined, both increased (CSK risk variant) and decreased (PTPN22 risk variant) B cell signaling has been reported [12,15]. However, these reports stand in contrast to older work suggesting that the B cells from SLE patients are hyper-responsive with increased anti-IgM- and -IgD-mediated [Ca2þ]i responses [16,17] and anti-IgM-induced protein tyrosine phosphorylation [17]. A potential resolution to this disparity lies in the observation that the B cells of SLE patients are subjected to high levels of pro-inflammatory cytokines, including Type I interferon (IFN) and BAFF, which could affect B cell signaling [18e21]. Thus, the impact of genetically determined differences in B cell signaling could be modulated in SLE patients as compared to those in healthy controls. Adding a further level of complexity to examination of signaling in SLE patients are the alterations in B cell homeostasis that are

http://dx.doi.org/10.1016/j.jaut.2015.01.009 0896-8411/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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N.-H. Chang et al. / Journal of Autoimmunity xxx (2015) 1e11

seen in this condition. SLE patients have altered proportions of plasma cells, pre-germinal center B cells, and memory B cells as compared to controls [22e29], which could affect the B cell activation threshold when the cell population is examined as a whole [29e31]. Additionally, SLE patients have a large proportion of B cells, even within the naïve B cell subset, that demonstrate changes consistent with prior activation in-vivo [23,28,29,32]. Thus, it is possible that the changes previously observed in SLE patients as compared to controls arise from these differences rather than from intrinsically altered B cell function. In this study, we have used Phosflow to contrast BCR signaling in well-defined peripheral blood B cell subsets of SLE patients and healthy controls. We show that the naïve B cells of SLE patients are indeed hyper-responsive to IgM receptor crosslinking. This heightened responsiveness is most marked in the transitional B cell subset, does not correlate with lupus risk variants that are proposed to alter BCR signaling, and is at least in part IFN-a-induced. Furthermore, the serum levels of IFN-a seen in SLE patients appear to be sufficient to modulate transitional B cell function in a way that could contribute to the breach of tolerance observed in this condition. 2. Materials and methods 2.1. Subjects SLE patients (n ¼ 39) were recruited from the University of Toronto Lupus Clinic. All patients satisfied 4 of the revised 1997 American College of Rheumatology classification criteria for SLE [33], were between the ages of 18 and 44 years (mean 33.88 ± 8.16) and taking 20 mg or less of prednisone per day (mean 8.43 ± 5.75 mg). The mean duration from diagnosis of SLE was 13.40 ± 7.05 years (range 1.80e31.88). Thirty (76%) of the patients were taking anti-malarial drugs and 25 (64%) were on immunosuppressive medications, which included: azathioprine (n ¼ 12), methotrexate (n ¼ 5), and mycophenolate mofetil (n ¼ 8). Disease activity was measured using the SLE Disease Activity Index (SLEDAI)-2K [34]. The mean SLEDAI-2K for the study patients was 3.97 ± 4.33, (range 0e20). Healthy controls (n ¼ 27) were between the ages of 18 and 44 years (mean 29.94 ± 7.29) with no family history of autoimmune disease. The study was approved by the Research Ethics Board of the University Health Network and all subjects provided informed consent. 2.2. B cell Phosflow PBMCs were isolated over a Ficoll (GE Healthcare) gradient, treated to remove residual RBCs, and washed twice at room temperature. The cells were resuspended in 5% FBS/RPMI (plus additives), rested at 37  C for 1 h, and then stimulated for 2 min with media alone, or 2 and 10 min with media containing 20 mg/ml of F(ab0 )2 goat anti-human IgM (Jackson ImmunoResearch). Following stimulation, the cells were fixed in 1% paraformaldehyde for 10 min at 37  C and then frozen at 80  C. After thawing, the cells were washed with PBS, and stained with various directly-conjugated Abs followed by Streptavidin-Pacific Blue™ (Invitrogen). Abs used for surface staining included: anti-IgD-FITC (IA6-2), -CD27allophycocyanin (L128), -CD19-allophycocyanin-H7 (HIB19), and -IgM-Biotin (G20-127) from BD Biosciences; and anti-CD38-PE-Cy7 (HIT2) from eBioscience. The cells were then permeabilized by incubation in 70% methanol on ice for 30 min. Following washing in PBS, the cells were resuspended in Perm/Wash™ buffer (BD Biosciences) and stained with the following Ab: anti-phospho (p)-SYKPE (pY348; 1120-722), -p-ERK1/2-PE (pT202/Y204; 20A), or -pPLCg2-PE (pY759; K86-689.37), all from BD Biosciences.

Approximately 1 million lymphoid events were acquired per sample, using an LSRII instrument (BD Biosciences) and were analyzed using Flow Jo software (TreeStar). 2.3. Measurement of Ca2þ mobilization B cells were enriched from PBMCs using RosetteSep (StemCell Technologies), serum-deprived for 1 h in Tyrode's buffer, and then labeled with 5 mM Indo-1 AM (Molecular Probes) and 0.03% pluronic F-127 (Molecular Probes) for 30 min at 37  C. After washing, the cells were stained with anti-CD20 (2H7), -CD27, -CD3 (HIT3a), -IgG (G18-145), -CD24 (ML5), and -CD38 Abs (all mAb BD Biosciences except CD38 eBioscience) and rested at 37  C for 10 min. Events were acquired for 1 min before addition of (Fab0 )2 goat antihuman IgM (20 mg/ml).

2.4. B cell co-culture with plasma Healthy control PBMCs (2  106 cells/ml) or purified B cells (0.4  106 cells/ml, isolated using a Human B cell Enrichment Kit, StemCell Technologies), were incubated for 1 h in 5% FBS/RPMI (plus additives) and various concentrations of IFN-a (PBL Interferon Source) or 50% plasma from healthy controls or SLE patients. For blocking studies, 1 mg/ml of neutralizing anti-human IFN-a (MMHA-2, PBL Interferon Source, sufficient to block 50 units IFN-a) or purified isotype-matched mouse IgG1 (MOPC-31C, BD Biosciences) was added. In some experiments, plasma was treated with RNase (20 mg/ml, Thermo Scientific) at 37  C for one hour or depleted of IgG by incubation with Protein G Sepharose gel (GE Healthcare) for one hour at 4  C.

2.5. B cell functional assays Naïve B cells were enriched from PBMCs using immunomagnetic beads (Naïve B cell isolation kit, Miltenyi Biotec). For measurement of B cell apoptosis, 1  106 PBMCs (for healthy control cells co-cultured with IFN-a) or 1  105 enriched naïve B cells (for SLE patients) were cultured overnight in medium supplemented with avidin alone (20 mg/ml, Sigma) together with biotinylated anti-IgM F(ab0 )2 (10 mg/ml, Jackson ImmunoResearch). For B cell proliferation experiments, cells were labeled with CFSE (Molecular Probes) and cultured for 3 days in medium alone or supplemented with anti-IgM F(ab0 )2 (10 mg/ml). Following culture, cells were stained with anti-CD19, -IgD, -CD27, -CD24, and -CD38 Abs to enable gating of specific B cell subsets. The proportion of apoptotic cells was determined by Annexin V-FITC (BD Biosciences) staining and the proportion of proliferating cells was assessed by quantifying the %CFSElo cells above background without stimulation.

2.6. Measurement of SYK protein expression B cells were isolated using RosetteSep, lysed in sample buffer, separated on a 10% SDS-PAGE gel, and transferred to a polyvinylidene difluoride membrane. Following blocking with 5% nonfat milk for 1 h, SYK was detected using a polyclonal rabbit anti-SYK Ab (1:2000, Cell Signaling) followed by HRP-conjugated anti-rabbit IgG Ab (1:1000, Cell Signaling). ACTIN levels were detected using a rabbit anti-ACTIN Ab (1:5000; AC-40, Sigma). Bound antibodies were detected using ECL Western Blotting Detection Reagent (Amersham), with the resultant bands being imaged with Kodak film and quantified using a Luminescent Image Analyzer (LAS3000; FUJIFILM).

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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2.7. SNP genotyping SNPs in BLK (rs2618476), LYN (rs7829816), PTPN22 (rs2476601), and CSK (rs34933034) were genotyped using Taqman SNP genotyping assays (Applied Biosystems). 2.8. Assessment of BAFF mRNA expression Total RNA was isolated from PAXgene tubes and converted to cDNA, as previously described [35]. qRT-PCR amplification was performed using TaqMan primers for BAFF (Assay identification Hs00198106_m1) and normalized to GAPDH expression [35]. 2.9. Statistical analysis The ManneWhitney non-parametric U test was used for comparisons between patients and controls. For analysis of correlations, a linear regression analysis was performed using Prism 5.0 software. P values of <0.05 were considered statistically significant. 3. Results 3.1. Optimization of a protocol to measure phosphorylated signaling molecules in distinct B cell subsets by flow cytometry Given the marked differences in the proportions and background activation levels of various B cell subsets in SLE patients as compared to healthy controls, it was necessary to develop a Phosflow protocol that could control for these differences. Instead of 90% methanol, we used 70% methanol as it preserved cell surface

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staining for many mAbs without a noticeable loss of the ability to stain intracellular signaling substrates. Staining with anti-CD19, -CD27, -IgM, -IgD (using a polyclonal reagent), and -CD38, but not -CD24, -CD10, or -CD86, was preserved under these conditions. This combination of stains permitted identification of naïve B cells as CD19þCD27IgDþCD38int/hi and allowed us to gate on subpopulations of B cells based on their IgM cell surface expression (Fig. 1A). It was necessary to control for B cell surface IgM levels because these are modulated by engagement with self-antigens invivo, affecting the capacity of B cells to signal following IgM crosslinking (also seen in Fig. 1B) [36,37]. Furthermore, we have shown that the proportion of IgMlo/ cells, which has been proposed to represent an anergic self-reactive B cell subset [38], is expanded in SLE [23]. By gating on IgDþCD38int/hi cells, we were also able to control for several CD27lo/ memory B cell populations that are expanded in SLE including a IgMIgD class-switched population [39], a CD21CD19hiCD38CD24IgDþ/IgMþ/ population [28], and a CD38CD19hiCD21þ/ population [29]. Following stimulation with F(ab0 )2 goat anti-human IgM and permeablization, the cells were stained intracellularly for expression of p-SYK, pPLCg2, or p-ERK. As shown in Fig. 1C, the maximal level of phosphorylation was seen at 2 min and this was significantly attenuated by 10 min. 3.2. Increased levels of p-SYK following IgM crosslinking in SLE B cells Since the naïve B cells of SLE patients express elevated levels of co-stimulatory molecules, suggesting prior activation in-vivo [23,40,41], we controlled for potential differences in basal

Fig. 1. Assessment of the levels of phosphorylated signaling intermediates in B cell subsets. (A) Contour plots showing the regions used to gate the naïve B cell subset in SLE patients and healthy controls. PBMCs were stained with anti-CD19, -IgD,- CD27, -IgM, and -CD38 Abs and naïve B cells gated at CD19þIgDþCD27. Regions used to gate IgMlo/ and IgMhi/int B cell subsets are shown, with IgMlo/ levels being established based upon staining of non-B cell populations. (B) Representative contour plots showing the levels of the indicated phosphorylated signaling intermediates for a representative healthy control and SLE patient. PBMCs were rested for 1 h at 37  C and then incubated for 2 min with media alone (top set of plots) or media containing 20 mg/ml of F(ab0 )2 goat anti-human IgM (bottom set of plots). Following fixation, naïve B cells were stained and gated as in (A), permeabilized and then stained intracellularly with anti-p-SYK, -p-PLCg2, or -p-ERK Abs. The regions used to determine the proportion of cells expressing elevated levels of phosphorylated signaling molecules were determined by setting a baseline of <5% positive cells for the total naïve B cell population of the lowest un-stimulated healthy control in each experiment. Numbers at the top left and right of each plot represent the percent positive cells for the IgMlo/ and IgMhi/int populations, respectively. (C) Graphs showing phosphorylation kinetics following IgM crosslinking for the total naïve B cell population. Shown are results for 1 healthy control (open circles) and 2 representative SLE patients (closed circles).

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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activation by including at least one healthy control in every experiment. The levels of phosphorylated signaling molecules in the lowest of the healthy controls in each experiment were considered to represent the baseline, with those in the other controls and SLE patients being expressed as a percentage using this baseline. Consistent with prior activation in-vivo, there was a trend to increased proportions of naïve B cells with high basal levels of phosphorylated signaling molecules in SLE patients as compared to controls (Fig. 2A), which achieved statistical significance for p-SYK and p-ERK. In general, the basal levels of phosphorylated signaling molecules correlated with each other, with all but the correlation between p-SYK and p-ERK in SLE patients achieving statistical significance (Fig. 2B and data not shown). There was a positive correlation between the proportions of p-SYKþ and CD86þ naïve B cells in SLE patients, whereas the levels of these cell populations were low in healthy controls and showed no association with each other (Fig. 2C). Notably, the elevated levels of basal phosphorylated signaling molecules in the naïve B cell population of SLE patients were not due to contamination with memory cell populations, such as the CD27CD38CD19hi subset that is reported to have high basal levels of p-SYK [29], as this population was excluded by our gating on CD38þ/þþ cells. Although we have previously noted a weak positive correlation between disease activity, as measured by the SLEDAI-2K, and costimulatory molecule expression in the naïve B cell compartment of SLE patients [23], there was no correlation between the basal levels of B cell signaling molecule phosphorylation and disease activity or treatment. To control for the basal differences in B cell activation between SLE patients and controls, results for stimulated cells were expressed as a percentage above their basal un-stimulated activation. As shown in Fig. 3A, the naïve B cell subset of SLE patients demonstrated significantly increased proportions of p-SYKþ cells, and trends to increased proportions of p-PLCg2þ and p-ERKþ cells at 2 and 10 min following IgM crosslinking as compared to controls. These increases were largely restricted to the IgMint/hi compartment. Differences in the percent positive cells in these populations reflected an overall shift in the levels of phosphorylation of the various populations (see Fig. 1B) and did not arise simply from elevated levels of SYK in SLE patients, since the levels of SYK in SLE patients and controls were similar when compared by Western Blot (Fig. 3B). As seen for basal cellular activation, there was a significant association between the levels of p-SYKþ and p-PLCg2þ, but not pERKþ cells following IgM crosslinking of naïve B cells from SLE patients, and similar findings were observed for healthy controls (Fig. 3C). No correlation was noted between the basal levels of these phosphorylated signaling molecules in media alone and the levels above background following IgM crosslinking (data not shown). Nor was there a correlation between the levels of phosphorylated signaling molecules following IgM crosslinking in SLE patients and disease activity or treatment.

cell subset with anti-CD24 and -CD10 under unfixed conditions confirmed that these cells were transitional cells (data not shown). Basal levels of p-SYK in each of the gated populations showed a similar trend to increased levels in SLE patients as compared to controls (Fig. 4B). Following IgM receptor crosslinking, the

3.3. Transitional B cells demonstrate the most marked increase in expression of p-SYK following IgM crosslinking

Fig. 2. Increased basal levels of phosphorylated signaling molecules in the naïve B cell subset of SLE patients. (A) Basal levels of p-SYK, p-PLCg2, and p-ERK in total (IgM), IgMlo/ and IgMhi/int naïve B cell subsets of SLE patients (filled circles, n ¼ 26) and healthy controls (open circles, n ¼ 16). Cells were incubated, stained, and gated as outlined in Fig. 1. Horizontal lines indicate the mean for each population examined. P values for the difference between controls and SLE patients were calculated using the ManneWhitney test. Statistically significant p values <0.05 (*) or <0.005 (**) are indicated. (B) Linear regression analysis comparing the proportion of phosphorylated cells between the different signaling intermediates examined for the total naïve B cell population of healthy controls and SLE patients. (C) Linear regression analysis showing the correlation between the proportion of naïve B cells in healthy controls and SLE patients with elevated basal levels of p-SYK and the proportion of freshly isolated denovo cells within the same population expressing elevated levels of CD86.

To investigate which cell populations within the IgMint/hi B cell subset were hyper-responsive to IgM receptor crosslinking, cells within the CD19þCD27IgDþ naïve subset were divided into mature (CD38int) and transitional (CD38hi) subpopulations and then stratified based upon their IgM cell surface expression (Fig. 4A). We were unable to gate transitional B cells as CD24hi and/ or CD10þ because staining with these mAbs was not preserved with our protocol. However, staining of the CD19þCD27IgDþCD38hi B

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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Fig. 3. Enhanced upregulation of p-SYK following IgM crosslinking of the naïve B cell population of SLE patients. (A) Percent cells demonstrating upregulation of the indicated phosphorylated signaling intermediates following IgM crosslinking. Freshly prepared PBMCs were rested for 1 h and then stimulated at 37  C for 2 min or 10 min with F(ab0 )2 antiIgM. Cells were stained and gated as outlined in Fig. 1, with the proportion of cells expressing phosphorylated signaling molecules in media alone being subtracted. Each circle represents the determination from an independent subject with open circles indicating healthy controls (n ¼ 16) and filled circles indicating SLE patients (n ¼ 26). Horizontal lines show the mean for each population examined. P values were calculated for the difference between healthy controls and SLE patients using the ManneWhitney test. Statistically significant p values, < 0.05 (*), <0.005 (**), or <0.0005 (***), are indicated. (B) Western blot showing similar basal levels of SYK in the B cells of healthy controls and SLE patients. The ratio of SYK to ACTIN (used as a loading control) is shown at the bottom of the figure, with each column representing an individual. (C) Linear regression analysis comparing the proportion of phosphorylated cells for the different signaling intermediates following IgM crosslinking. Results shown are for the total naïve B cell population of healthy controls and SLE patients.

proportion of p-SYKþ cells was increased in SLE patients as compared to controls for the majority of subpopulations at 2 and 10 min (Fig. 4C). This increase was most marked for the IgMhi transitional cells and was seen for both transitional 1 and 2 cell populations (Fig. 4D). As IgMint cells may be partially antigen-engaged [36,37], we focused our subsequent analysis on the IgMhi compartment, contrasting transitional and mature cells. Within these compartments, there were no significant differences between SLE patients and controls in the levels of cell surface IgM or CD19 that could account for the differences observed (mean MFI CD19 ± SD: CD38intIgMhi, controls ¼ 99.78 ± 175.52, patients ¼ 48.59 ± 169.99, p ¼ 0.36; CD38hiIgMhi subset, controls ¼ 63.33 ± 150.82, patients ¼ 19.72 ± 135.22, p ¼ 0.35; mean MFI IgM ± SD: CD38hiIgMhi, controls ¼ 18,368 ± 3564, patients ¼ 19,581 ± 6984; CD38intIgMhi, controls ¼ 17,366 ± 3083, patients ¼ 16,007 ± 5263; p ¼ 0.72 and 0.24 respectively). To determine whether the increased SYK phosphorylation in SLE patients is propagated to downstream signaling events, we examined the proportion of p-PLCg2þ and p-ERKþ cells in each

population and assessed the correlation between the proportions of p-SYKþ, and p-PLCg2þ or p-ERKþ cells. As seen for the global naïve B cell population, the proportions of p-PLCg2þ and p-ERKþ cells were not significantly increased in SLE patients as compared to healthy controls at 2 and 10 min following IgM receptor crosslinking for either IgMhi population (data not shown). Nevertheless, consistent with the findings for the global naïve B cell population, there was a moderate correlation between the proportion of pSYKþ and p-PLCg2þ cells in the mature and transitional IgMhi cell subsets of healthy controls or SLE patients (Fig. 4E), and transitional cells from SLE patients demonstrated increased Ca2þ mobilization as compared to those from controls (Fig. 4F), suggesting that the transitional B cell hyper-responsiveness is propagated to more downstream signaling events. 3.4. The levels of SYK phosphorylation fluctuate over time and are not correlated with specific genotypes Both genetic and pro-inflammatory factors could lead to the increased levels of p-SYK following IgM crosslinking in the SLE B

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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Fig. 4. Transitional B cells demonstrate the most marked increase in expression of p-SYK following IgM crosslinking. (A) Contour plots demonstrating representative gating of transitional (CD38hi) and mature (CD38int) naïve B cell subsets for a single healthy control and SLE patient. Plots are gated on CD19þIgDþCD27 naïve B cells as shown in Fig. 1A. The regions used to gate on IgMint and IgMhi cells are shown, and were determined by gating first on the predominantly IgMhi transitional B cell population. (B) Basal proportion and (C) proportion above un-stimulated background following IgM crosslinking of p-SYKþ cells in CD38int (mature) and CD38hi (transitional) B cell subsets stratified based on the levels of cell surface IgM for 26 SLE patients (filled circles) and 16 healthy controls (open circles). Cells were incubated and stained, as outlined in Fig. 1. (D) Proportion of p-SYKþ cells in the T1 (gated as CD38þþþIgMþþþ) and T2 (gated as CD38þþIgMþþ) subpopulations of the transitional B cell subset of healthy controls and SLE patients. Horizontal lines indicate the mean for each population examined. P values for the difference between controls and SLE patients were calculated using the ManneWhitney test. Statistically significant p values <0.05 (*), <0.005 (**) or <0.0005 (***) are indicated. (E) Linear regression plots showing the correlation between the proportion of p-SYKþ and p-PLCg2þ or p-ERKþ cells for IgMhi mature (CD38int) or transitional (CD38hi) populations. (F) Representative plots of intracellular calcium following IgM crosslinking of transitional B cells. Purified B cells were loaded with Indo-1 then surface stained. Following 1 min acquisition to establish a baseline, the cells were stimulated with 20 mg/ml of F(ab0 )2 anti-IgM (indicated by arrow). Transitional B cells were gated as CD24hiCD38hiIgGCD27CD20þCD3. Results from 2 independent experiments are shown with those for a representative SLE patient (thick line) overlaid onto a normal control (thin line) performed on the same day. The results are representative of 4 independent experiments.

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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cells. We reasoned that if genetic factors alone were associated with this phenotype, then the proportion of p-SYKþ cells following IgM crosslinking would be relatively stable over time. Therefore, six patients with elevated levels of p-SYKþ cells on their first visit were reassessed at a later visit (22e25 months later). As shown in Fig. 5A, the majority of these patients demonstrated changes in the levels of p-SYK which roughly paralleled changes in disease activity, raising the possibility that pro-inflammatory factors contribute to the altered B cell signaling in SLE.

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To determine whether pro-inflammatory factors could act in tandem with genetic factors to produce the SYK hyperphosphorylation phenotype, the association between this phenotype and lupus risk alleles for the B cell signaling molecules BLK, LYN, PTPN22, and CSK was examined [9,10,12e14]. Overall, relatively few of the patients were homozygous for these lupus risk alleles and the proportion of the patients heterozygous for the risk alleles ranged from 0 to 9.5%. As shown in Fig. 5B, the levels of SYK phosphorylation in IgMhi transitional B cells following IgM

Fig. 5. Pro-inflammatory factors rather than genetic differences lead to increased responses of transitional B cells in SLE patients. (A) Proportion of p-SYKþ cells following IgM crosslinking for 2 min in the total naïve B cell population of 6 SLE patients assessed at two independent visits spaced 22e25 months apart. Shown adjacent to the determination for the last visit are the SLEDAI-2K disease activity scores (first visit, last visit). (B) Lack of correlation between the proportion of p-SYKþ cells at 2 min IgM following crosslinking in the IgMhi transitional (CD38þþ) B cell subset of SLE patients (n ¼ 21) and lupus risk variants for selected B cell signaling genes. For each indicated SNP, individuals who are homozygous for the lupus risk variant are shown on the right of the graph (filled circles) with heterozygotes (half-filled circles) shown in the center. Lines indicate the median for each population. (C) Incubation of healthy control PBMCs with 50% SLE plasma, but not healthy control plasma, augments SYK phosphorylation of IgMhi transitional (CD38hi) cells following IgM crosslinking. PBMCs were rested, stimulated with media alone or containing (Fab0 )2 anti-IgM for 2 min, and stained as outlined in Fig. 1, except that healthy control or SLE plasma, at a final concentration of 50%, was present throughout the 1 h rest period and stimulation. Shown are results for incubation of healthy control cells with healthy control plasma (Control), and plasma samples from SLE patients who had lower levels (SLElo, range p-SYK ¼ 20.9e42.2%) or higher levels (SLEhi, range p-SYK ¼ 60.3e72.5%) of p-SYKþ cells following IgM crosslinking. Background levels of p-SYKþ cells in the absence of (Fab0 )2 anti-IgM have been subtracted and did not differ between healthy control and SLE plasma. (D) The increased phosphorylation of healthy control B cells induced by SLE plasma following IgM crosslinking is partially blocked by incubation with anti-IFN-a Ab. Experiments were carried out as outlined in (C), except that anti-human IFN-a or purified isotype-matched mouse IgG1 was added throughout the culture period. (E) The increased SYK phosphorylation seen with SLE patient plasma results from a direct action of plasma on B cells. PBMCs and purified B cells from the same healthy control were incubated with healthy control (open circles) or SLE (filled circles) plasma, as outlined in (C). (F) Effect of RNase or IgG-depletion (IgG) on the increased SYK phosphorylation induced by SLE patient plasma. P values for the differences between controls and SLE patients were calculated using the ManneWhitney test. Statistically significant p values <0.05 (*) or <0.005 (**) are indicated.

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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crosslinking were not different between SLE patients with and without the lupus risk alleles and similar findings were seen for the IgMhi naïve mature B cell subset (data not shown). Thus, polymorphisms in these risk alleles did not appear to be making a major contribution to the altered B cell function that was observed in our subset of SLE patients.

3.5. SLE patient plasma induces SYK hyper-phosphorylation upon IgM crosslinking which is blocked by anti-IFN-a Ab To further explore the possibility that circulating proinflammatory factor(s) induce the altered B cell function in SLE patients, plasma from SLE patients or healthy controls was coincubated with PBMCs from healthy controls. In preliminary experiments, it was found that incubation of PBMCs with plasma overnight (16 h) resulted in significant death of B cells. Therefore, PBMCs were incubated in the presence or absence of patient or control plasma for 1 h and then the cells were crosslinked with anti-IgM. Incubation of control B cells with SLE plasma, in particular plasma from patients who had previously been shown to have high proportions of p-SYKþ B cells following IgM crosslinking, induced a greater proportion of pSYKþ cells within the IgMhi transitional B cell subset than plasma from healthy controls (Fig. 5C). This induction required IgM receptor crosslinking as the levels of basal pSYK were similar for control and patient plasma. There were minimal increases in the proportion of pSYKþ cells in the mature IgMhi and transitional IgMint cell subsets under these conditions (data not shown). One of the central pro-inflammatory factors elaborated in SLE is Type I IFN, which has been shown to have pleomorphic effects on multiple immune populations including B cells [42]. We therefore examined whether the altered B cell function induced by the plasma from lupus patients could be blocked by incubation with an anti-IFN-a Ab. This treatment resulted in a significant reduction in the proportion of p-SYKþ cells following IgM crosslinking for cells incubated with SLE plasma, but not control plasma, as compared to an irrelevant mAb (Fig. 5D). Thus, a significant component of the altered SYK phosphorylation induced by SLE plasma appears to arise from the presence of IFN-a in the plasma. Purified B cells showed the same increased SYK phosphorylation with SLE patient plasma as PBMCs (Fig. 5E), consistent with the altered B cell function being the result of a direct effect of SLE patient plasma on B cells. Since SLE patients have increased levels of IgG immune complexes, we also assessed the effect of IgG depletion and RNase inhibition on the B cell hyper-responsiveness seen with SLE plasma. IgG depletion led to slightly increased proportions of p-SYKþ cells following IgM crosslinking, probably due to the inhibitory effect of IgG binding to FCgRIIB, whereas RNase had a general inhibitory effect on B cell activation (Fig. 5F). Nevertheless, for both conditions, SYK phosphorylation remained elevated following incubation with SLE plasma as compared to healthy control plasma, indicating that the B cell hyperresponsiveness induced by SLE patient plasma does not arise from the presence of immune complexes. BAFF has also been shown to enhance SYK phosphorylation [21]. Therefore, to determine whether BAFF could be contributing to the altered B cell function that was observed in SLE patients, we assessed the correlation between SYK phosphorylation and peripheral blood BAFF RNA expression for 21 patients who had RNA available. Although BAFF expression was significantly elevated in SLE patients, as compared to controls (p ¼ 0.0047), there was no correlation between the levels of BAFF in SLE patients and either basal or anti-IgM-induced phosphorylation for any of the signaling intermediates examined (data not shown).

3.6. IFN-a induces B cell signaling and functional abnormalities that recapitulate those observed for SLE transitional B cells We next sought to confirm that incubation of healthy control B cells with IFN-a induced the same signaling abnormality as observed with lupus plasma. Control PBMCs were incubated for 1 h with varying concentrations of IFN-a and then crosslinked with anti-IgM. There was minimal induction of SYK phosphorylation with IFN-a in the absence of IgM crosslinking; however, IFN-a significantly augmented SYK phosphorylation following IgM crosslinking in a dose-dependent fashion (Fig. 6A). As with the patient plasma, this effect was most pronounced in IgMhi transitional B cells and was also seen for purified B cells (Fig. 6B). Type I IFNs are reported to enhance B cell survival [43e46] and proliferation following BCR crosslinking [18,47,48]; however, it is unknown whether the function of the transitional subset is similarly affected. To address this question, naïve B cells were purified from healthy controls and hyper-crosslinked with biotinylatedF(ab0 )2 goat anti-human-IgM and avidin to induce apoptosis, or stimulated with F(ab0 )2 goat anti-human-IgM to induce proliferation, in the presence of various concentrations of IFN-a. As previously reported for the total B cell population, addition of IFN-a led to enhanced survival and proliferation of transitional B cells (Fig. 6C&D). Consistent with the possibility that IFN-a may be altering transitional B cell function in-vivo in SLE, there was a positive correlation between the proportion of p-SYKþ transitional B cells at 2 min following IgM receptor crosslinking, and transitional B survival and proliferation in SLE patients (Fig. 6E&F) but not controls (data not shown). 4. Discussion Increased B cell responses to BCR crosslinking have been noted in SLE for many years [16,17]. However, the immune mechanisms leading to this observation have been difficult to dissect due to limitations in the techniques used to assay B cell function, which precluded analysis of individual B cell populations. This problem was further compounded by the marked differences in the proportion or activation status of various peripheral B cell subsets between SLE patients and controls [22e28,40,41], which prevented definitive attribution of the differences observed to disturbances of function rather than changes in B cell subsets. In this study, we have overcome these problems by using Phosflow. We show that even when we control for IgM cell surface expression and developmental stage, IgM receptor crosslinking of naïve B cells leads to enhanced phosphorylation of downstream signaling molecules, in particular SYK, in lupus patients. Thus, the signaling differences observed between lupus patients and controls arise from altered signaling and not simply from the altered distribution of various B cell subsets in lupus patients. The observation that the SYK hyper-phosphorylation phenotype fluctuated in some patients between visits suggested that this abnormality did not arise solely from genetic polymorphisms that lead to enhanced B cell signaling. Consistent with this finding, none of the lupus-associated SNPs in several B cell signaling molecules, whose function was predicted to affect SYK phosphorylation following BCR crosslinking, were associated with the hyperphosphorylation phenotype. In further support of a role for proinflammatory factors in the generation of this phenotype, incubation of healthy control cells with plasma from lupus patients recapitulated the phenotype. Several findings suggest that one of the major pro-inflammatory factors that generates this phenotype is IFN-a: 1) IFN-dependent phenotypes observed in-vitro recapitulated those seen for freshly isolated ex-vivo cells from lupus patients; 2) Incubation of control B cells with IFN-a or lupus plasma

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N.-H. Chang et al. / Journal of Autoimmunity xxx (2015) 1e11

Fig. 6. IFN-a alters B cell function in a manner consistent with that observed in SLE patients. Incubation of healthy control (A) PBMCs or (B) purified B cells with IFN-a enhances SYK phosphorylation following IgM crosslinking. Cells were rested in the presence of varying concentrations of IFN-a for 1 h and then incubated with media alone (dashed lines) or containing (Fab0 )2 anti-IgM for 2 min (solid lines). The cells were then fixed and stained as outlined in Fig. 1, and gated as shown in Fig. 4. (A) shows results for 3 different sets of healthy control cells, whereas (B) shows the comparison between PBMCs (open circles) and purified B cells (filled circles) from the same healthy control. Results in the left panel are for IgMhi transitional (CD38hi) cells and in the right panel are for IgMhi mature (CD38int) naïve cells. (C) Incubation of healthy control transitional B cells with IFN-a leads to enhanced survival of transitional B cells. PBMCs were cultured overnight with avidin alone or avidin þ biotinylated (Fab0 )2 anti-IgM. Apoptosis was quantified by Annexin V staining. The percent surviving cells was calculated as the surviving cells in stimuli (avidin þ biotinylated (Fab0 )2 anti-IgM) divided by the surviving cells in avidin alone. Transitional B cells were gated as CD19þIgDþCD27CD24hiCD38hi. Results are shown for three independent experiments. (D) Incubation of healthy control transitional B cells with IFN-a augments anti-IgM induced proliferation. PBMCs were labeled with CFSE and cultured for 3 days in medium alone or supplemented with (Fab0 )2 anti-IgM. Transitional B cells were gated as CD19þIgDþCD27CD24hiCD38hi, with similar proportions seen for un-stimulated and stimulated conditions. Results are expressed as % CFSElo and are representative of three independent experiments. (E) Survival of transitional B cells correlates with SYK phosphorylation following IgM crosslinking in SLE patients (n ¼ 13). Enriched naïve B cells were cultured with avidin alone or together with biotinylated (Fab0 )2 anti-IgM overnight. The percent surviving cells was calculated the same way as in (B). (F) The

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produced remarkably similar signaling phenotypes; and 3) Addition of anti-IFN-a Ab to lupus, but not control, plasma resulted in significant reduction in the levels of SYK phosphorylation in coculture experiments with control B cells. We recognize that incubation of B cells for 1 h with IFN-a or patient plasma resulted in fairly modest changes as compared to those observed for freshly isolated B cells from SLE patients. This is most likely due to the relatively short duration of incubation which does not precisely recapitulate the in-vivo situation where the cells are continuously bathed in pro-inflammatory factors. In support of this concept, incubation of control cells with IFN-a for 4 h resulted in significant augmentation of anti-IgM-induced p-SYK, as compared to that seen for 1 h (data not shown). Nevertheless, we cannot exclude a role for other pro-inflammatory factors in-vivo in the generation of the SYK hyper-phosphorylation phenotype in some patients. In this connection, BAFF has recently been shown to promote SYK phosphorylation in a BAFF receptor-dependent fashion [21]. While we did not see a correlation between BAFF RNA levels and the SYK hyper-phosphorylation phenotype, it is still possible that elevated levels of BAFF contribute to some of the B cell hyper-responsiveness that is observed, particularly for those patients where anti-IFN-a Ab did not reduce the levels of p-SYK induced by SLE patient plasma to the levels seen for control plasma. Although previous work has demonstrated that incubation of B cells with Type I IFN can alter their function, the impact of Type I IFN on proximal signaling events following IgM receptor crosslinking has not been reported nor has a direct link been made between the B cell hyper-responsiveness and the high levels of IFN seen in lupus patients. In mice, Type I IFN has been shown to lead to increased Ca2þ mobilization [18]. Given that the levels of p-SYK are strongly correlated with p-PLCg2 following IgM receptor crosslinking in the mature B cell population of lupus patients and that pPLCg2 is immediately upstream of Ca2þ mobilization in mature B cells, it is likely that IFN-induced signaling changes contribute to the increased Ca2þ mobilization observed in lupus patients. Recently, Wu et al. found reduced levels of PTEN and elevated levels of miR-7, a negative regulator of PTEN expression, in the B cells of lupus patients [32]. They further demonstrated that a miR-7 antagomir reduced the augmented Ca2þ mobilization seen in SLE B cells following IgM crosslinking and enhanced expression of PTEN, suggesting that the B cell hyper-responsiveness in SLE patients may be partially related to defective regulation of PTEN. As PTEN plays an important role in the negative regulation of the PI3K signaling pathway, which is downstream of SYK, it is possible that the enhanced generation of p-SYK induced by IFN-a following BCR crosslinking acts in tandem with reduced levels of PTEN in SLE patients to lead to B cell hyper-responsiveness. This concept is supported by previous work implicating the PI3K signaling pathway in the rescue of B cells from apoptosis by Type I IFN [43,44]. It is not known whether IFN-a induces expression of miR-7 in B cells and thus could play a role in both mechanisms of B cell hyper-responsiveness in SLE. The proximal signaling events leading to increased generation of p-SYK following IgM receptor crosslinking in the presence of IFN-a are also not clear. IFN-a binds to the IFN-a receptor as well as CD21, and signaling through both of these receptors has been shown to lead to upregulation of IFN-induced gene expression [42,49]. It is

proportion of proliferating transitional B cells correlates with SYK phosphorylation following IgM crosslinking in SLE patients (n ¼ 18). Enriched naïve B cells were labeled with CFSE and cultured for 3 days in medium alone or supplemented with (Fab0 )2 antiIgM. Transitional B cells were gated as CD19þIgDþCD27CD24hiCD38hi, with similar proportions seen for un-stimulated and stimulated conditions. Results are expressed as % CFSElo above background with media alone.

Please cite this article in press as: Chang N-H, et al., Interferon-a induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.01.009

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possible that interaction between IFN-a and CD21 leads to enhanced phosphorylation of LYN, which in turn promotes SYK phosphorylation. Alternatively, signaling through the IFN-a receptor could indirectly mediate SYK phosphorylation, as a wide range of receptors, which lack ITAM motifs but act through upstream adapters, have been shown to lead to SYK phosphorylation [50]. In addition to enhancing Ca2þ mobilization following IgM crosslinking, incubation of B cells with Type I IFN augments B cell proliferation [47,48] and protects B cells from spontaneous and IgM- or Fas-mediated apoptosis [43e46]. As observed previously for mature B cells, incubation of control B cells with IFN-a led to increased survival and proliferation of the transitional B cell subset. The correlation between the levels of p-SYK, and survival and proliferation following IgM crosslinking of the transitional B cell subset of lupus patients suggests that the elevated levels of IFN observed in lupus are affecting transitional B cell function in-vivo. Transitional B cells are more sensitive to apoptosis and undergo less proliferation in response to IgM receptor crosslinking compared to mature naïve B cells [51]. These functional differences are thought to play an important role in the maintenance of B cell tolerance, as a significant proportion of the self-reactive cells within the transitional B cell population are purged in a BAFF-dependent fashion prior to entry into the mature B cell compartment [52]. The findings reported herein suggest that the elevated levels of IFN-a in lupus patients alter transitional B cell function in a way that may lead to increased survival and/or proliferation of these self-reactive B cells. Since IFN-a has also been shown to induce BAFF [53], the elevated levels of IFN-a observed in lupus patients may act both directly and indirectly to enhance survival of self-reactive B cells contributing to the breach of tolerance in this condition. 5. Conclusions Naïve B cells, particularly in the transitional subset, demonstrate enhanced SYK phosphorylation following IgM receptor crosslinking in SLE. This hyper-responsiveness is induced, at least in part, by the high levels of IFN-a in SLE patients' blood, which also lead to disturbances of transitional B cell function that could contribute to the breach of B cell tolerance in this condition. Acknowledgments This study was supported by grants from CIHR (FRN# 93766) and the Arthritis Research Foundation of UHN (Edward Dunlop Research Challenge Grant). C.L-M. was supported by a ClinicianScientist Salary Award from the Arthritis Research Foundation of UHN. P.R.F. was the recipient of a Distinguished Senior Investigator Award from The Arthritis Society. J.E.W. was supported by the Arthritis Centre of Excellence and the Arthritis Research Foundation of UHN. References [1] Hibbs ML, Tarlinton DM, Armes J, Grail D, Hodgson G, Maglitto R, et al. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell 1995;83:301e11. [2] Majeti R, Xu Z, Parslow TG, Olson JL, Daikh DI, Killeen N, et al. An inactivating point mutation in the inhibitory wedge of CD45 causes lymphoproliferation and autoimmunity. Cell 2000;103:1059e70. [3] Nishimura H, Nose M, Hiai H, Minato N, Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999;11:141e51. [4] Nishizumi H, Taniuchi I, Yamanashi Y, Kitamura D, Ilic D, Mori S, et al. Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice. Immunity 1995;3:549e60. [5] Pan C, Baumgarth N, Parnes JR. CD72-deficient mice reveal nonredundant roles of CD72 in B cell development and activation. Immunity 1999;11: 495e506.

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