Disturbed B cell homeostasis in newly diagnosed giant cell arteritis and polymyalgia rheumatica.

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Arthritis & Rheumatism DOI 10.1002/art.38625

Running title: B cells in GCA and PMR

Disturbed B cell Homeostasis in Patients with Newly-diagnosed Giant Cell Arteritis and Polymyalgia Rheumatica Kornelis S. M. van der Geest1, Wayel H. Abdulahad1, Paulina Chalan1, Abraham Rutgers1, Gerda Horst1, Minke G. Huitema1, Mirjam P. Roffel1, Caroline Roozendaal2, Philip M. Kluin3, Nicolaas A. Bos1, Annemieke M. H. Boots1†, Elisabeth Brouwer1† 1

Kornelis van der Geest, MD; Wayel Abdulahad, PhD; Paulina Chalan, MS; Abraham Rutgers, MD/PhD; Gerda Horst, BSc; Minke Huitema, BSc; Mirjam Roffel, BSc; Nico Bos, PhD; Annemieke Boots, PhD; Elisabeth Brouwer, MD/PhD. Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. 2

Caroline Roozendaal, PhD. Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. 3

Philip Kluin, MD/PhD. Department of Pathology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

† AMH Boots and E Brouwer contributed equally to this study as senior authors.

Correspondence to: Elisabeth Brouwer, MD/PhD, Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, Hanzeplein 1, 9700RB, The Netherlands, [email protected].

Key words: giant cell arteritis, polymyalgia rheumatica, B lymphocytes, regulatory B lymphocytes, BAFF, IL-6, monocytes

Word count: 250 words (abstract), 4016 words (manuscript), 25 pages, 48 references, 6 figures + 2 supplementary files.

Financial support: This work was supported by grants from MSD and the Dutch Arthritis Association (NC Smit Fonds, Reumafonds grant no. 9-1-201).

Disclosures: AMH Boots is a former employee of Organon NV, now Merck Research Laboratories, MSD. The other authors have nothing to disclose.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.38625 © 2014 American College of Rheumatology Received: Jul 19, 2013; Revised: Jan 29, 2014; Accepted: Mar 06, 2014

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Abstract Objective Several lines of evidence indicate that B cells may be involved in the immunopathology of giant cell arteritis (GCA) and polymyalgia rheumatica (PMR). We aimed to study the distribution of defined B cell subsets, including Beffector cells and Bregulatory cells, in GCA and PMR patients before and after corticosteroid treatment. Methods Circulating B cells were analyzed in 34 newly-diagnosed, non-treated patients with GCA and PMR, and in 44 follow-up samples of GCA and PMR patients receiving corticosteroids for 2 weeks and 3 months. For comparison, 40 age-matched, healthy controls (HCs) and 11 rheumatoid arthritis (RA) patients were included. Serum BAFF levels were determined and temporal arteries were studied by immunohistochemistry. Results Newly-diagnosed GCA and PMR patients, but not RA patients, had decreased numbers of circulating B cells compared to HCs. B cell numbers recovered rapidly in treated GCA and PMR patients in remission. This recovery was not achieved by compensatory hyperproliferation or enhanced bone marrow production. B cell numbers inversely correlated with ESR, CRP and serum BAFF. TNF-α+ Beffector cells but not IL-10+ Bregulatory cells were decreased in newly diagnosed GCA and PMR patients. Following treatment, circulating numbers of Beffector cells normalized. The returning Beffector cells demonstrated an enhanced capacity to produce IL-6. Few B cells were found in temporal artery biopsies of GCA patients. Conclusions We show for the first time that the distribution of B cells is highly disturbed in GCA and PMR and that B cells likely contribute to the enhanced IL-6 response in both diseases.

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Introduction Giant cell arteritis (GCA) is the most common type of primary vasculitis in Western countries (1). GCA is characterized by inflammation of medium-sized and large arteries resulting in classic symptoms like headache, blindness and stroke (2). Polymyalgia rheumatica (PMR) is a closely-related disorder associated with tenderness and stiffness of hips and shoulders. Corticosteroids are the cornerstone of treatment in GCA and PMR, but unfortunately come with considerable side effects (3). Insight into the pathogenesis of GCA and PMR is crucial for the development of targeted therapies in both diseases. Current evidence suggests that dendritic cells, T cells and macrophages are important players in GCA and PMR (4, 5). Although B cells are neither predominant cells in inflamed temporal arteries of GCA patients nor in affected joints of PMR patients (5-10), several findings indicate that B cells are not mere bystanders in GCA and PMR. General B cell activation in GCA and PMR is suggested by the increased presence of auto-antibodies against various endogenous antigens in sera of GCA and PMR patients (11-15). Furthermore, early studies have indicated that deposits of immunoglobulins are present in inflamed temporal arteries of GCA patients (16, 17). Scarce data on B cell depletion therapy in GCA suggest that this treatment may ameliorate GCA disease activity (18), as previously described in small vessel vasculitis and Takayasu arteritis (19, 20). Currently, little is known about B cells and their distribution in GCA and PMR patients. Emerging data indicate that B cells not only produce antibodies, but also modulate T cell responses by secreting pro- and anti-inflammatory cytokines such as TNF-α and IL-10, respectively (21-23). Furthermore, Beffector cells can potentiate T cell-mediated autoimmunity via secretion of IL-6 (24). So far, the contribution of Beffector cells and Bregulatory cells to GCA and PMR has not been studied. We hypothesized that B cells may be involved in the

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immunopathology of GCA and PMR. Therefore, we investigated the distribution of defined B cell subsets in GCA and PMR patients before and after corticosteroid treatment.

Patients and Methods Study population In a prospective study, 34 newly-diagnosed patients with GCA (n=16) and PMR only (n=18) were consecutively enrolled (Supplemental table 1). None of the patients received corticosteroids or disease-modifying antirheumatic drugs (DMARDs) at the time of blood withdrawal. GCA and PMR patients fulfilled the ACR and Chuang/Hunder criteria, respectively (25, 26). In GCA patients, a 18F-fluorodeoxyglucose (FDG)-PET/CT scan with similar or enhanced glucose uptake by large vessels in comparison to the liver was considered an equally diagnostic criterion as a positive temporal artery biopsy. We precluded atherosclerosis as main cause for this glucose uptake by co-examining the vessels for calcifications. In total, 15 out of 16 GCA patients had a positive temporal artery biopsy and/or demonstrated evidence for GCA on the FDG-PET/CT scan. We collected 44 follow-up samples of GCA and PMR patients, who were in remission after 2 weeks and 3 months of corticosteroid treatment. Remission was defined as absence of symptoms and a normal ESR (< 30 mm/hr). We also collected blood samples from 8 GCA and PMR patients who developed a relapse during follow-up. Relapse was defined as an increase of ESR from normal to 30 mm/hr or greater, plus at least one symptom of active GCA or PMR not attributable to other causes. We obtained blood samples from 40 age-matched, healthy controls that were thoroughly screened for past or actual morbidities. As inflammatory disease controls, we also included 11 newly-diagnosed, corticosteroid/DMARD-free patients with rheumatoid arthritis (RA). The study was approved by the institutional review board of the UMCG and all study participants gave written informed consent.

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Treatment GCA patients and PMR patients were initially treated with 60 mg/day and 20 mg/day of prednisolone, respectively. Tapering of corticosteroid treatment was started after 3-4 weeks and further continued based on clinical and laboratory findings during follow-up. After 3 months, the median prednisolone dose was 20 mg/day (range 15-40) in GCA patients and 15 mg/day (range 7.5-17.5) in PMR patients. Quantification of B cells and B cell differentiation subsets Absolute numbers of CD19+ B cells were determined according to the MultiTest TruCount method (BD), as described by the manufacturer. Data were acquired on a FACSCanto-II (BD) and analyzed with FACSCanto Clinical Software (BD). In addition, mononuclear cells were isolated from heparinized blood with Lymphoprep (Axis-Shield). Isolated cells were stained with the following monoclonal antibodies: CD19-efluor605, CD24-PcP-efluor710, CD24-PE, CD27-APC-efluor780, CD38-PE-Cy7 (all eBioscience), IgD-V450 and IgM-APC (BD). To perform intracellular staining with monoclonal antibodies Ki-67-PcP-Cy5.5 and Bcl-2-FITC (BD) the cells were first treated with a FoxP3 staining buffer set (eBioscience). Samples were measured on a LSR-II (BD) and analyzed with Kaluza Flow Analysis Software (Beckman Coulter). B cell differentiation subsets were gated as previously described (27-29). Intracellular cytokine staining Whole blood samples were diluted 1:1 with RPMI and stimulated with PMA and calcium ionophore A23187, or with different concentrations of recombinant human BAFF (Enzo Life Sciences) in the presence of brefeldin A (BFA, Sigma) during 4 hours. After red blood cell lysis with ammonium chloride, stimulated cells were treated with Fix/Perm reagent A and B (Invitrogen) and stained with the following antibodies: CD19-APC-efluor780, CD3-efluor605, CD4-efluor450, IL-6-APC (all eBioscience), CD22-FITC, TNF-α-PE, IL-10-APC (BD) and CD14-PE, TNF-α-PcP-Cy5.5 (Biolegend).

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ELISA Serum levels of BAFF were measured with an ELISA kit from R&D Systems. Measurements were performed with a Versamax microplate-spectrophotometer from Molecular Devices. Immunohistochemistry Temporal artery biopsies were obtained from 6 GCA patients not receiving corticosteroid treatment and 3 controls with non-inflamed temporal artery biopsies. The tissue was fixed in 10% neutral buffered formalin and paraffin embedded. Sections of 5 µm thickness were deparaffinised in xylene and rehydrated in graded ethanol. Antigens were retrieved and endogenous peroxidase was blocked. Staining was performed with antibodies from Dako against CD3 (clone F7.2.38), CD20 (L26) and CD138 (MI15) and visualized with DAB. Semi-quantitatively scoring of CD3, CD20 and CD138 expressing cells was performed by two independent investigators on a five-point scale (0-4): 0 = no positive cells, 1 = occasional positive cells, 2 = moderate number of positive cells, 3 = large number of positive cells, 4 = very large number of positive cells. The scores of both investigators were averaged. Statistical analysis HCs and patient groups were first analyzed with the Kruskall-Wallis test. The Mann Whitney U test was used to compare the different patient groups with healthy controls. Analyses were performed with GraphPad Prism 5.0. Correlations were assessed using the Spearman’s rank correlation coefficient. Two –tailed p values less than 0.05 were considered significant.

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Results Decreased numbers of circulating B cells in GCA and PMR To study whether B cells are modulated in GCA and PMR, we first determined absolute numbers of CD19+ B cells in peripheral blood of untreated, newly-diagnosed GCA (nGCA) and PMR (nPMR) patients (Fig.1A). Numbers of circulating B cells were clearly decreased in nGCA and nPMR patients when compared to healthy controls (HCs). In contrast, peripheral B cell numbers were normal in newly-diagnosed RA (nRA) patients. To study the effect of corticosteroid treatment, absolute numbers of CD19+ B cells were determined after 2 weeks and 3 months of treatment in GCA and PMR patients in remission (rGCA and rPMR). Intriguingly, two weeks of corticosteroid treatment already led to normalization of circulating B cell numbers in rGCA and rPMR patients (Fig.1B and 1C). After 3 months of treatment, B cell numbers were retained in rPMR patients, but further increased in rGCA patients. Interestingly, B cell numbers dropped again when corticosteroid treated GCA and PMR patients developed a relapse (Fig.1A). Since B cell numbers seemed to correlate inversely with disease activity in GCA and PMR patients, we tested whether circulating B cell numbers in patients also inversely correlated with systemic inflammatory markers. Indeed, numbers of circulating B cells inversely correlated with ESR (Fig.1D) and CRP (Spearman’s rho -0.53, p < 0.001) in GCA and PMR patients. B cell counts in GCA patients did not correlate with the number of inflamed large arteries on the FDG-PET/CT scan (data not shown). Disturbed numbers of B cell differentiation subsets in GCA and PMR To identify whether particular B cell differentiation subsets were modulated in nGCA and nPMR patients, we further delineated B cells based on expression of B cell differentiation markers, as shown in figures 2A-B. In essence, nearly all B cell differentiation subsets were decreased in nGCA and nPMR patients when compared to HCs (Fig.2C). Unswitched memory

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B cells were an exception, as these cells were increased in nGCA patients, and unchanged in nPMR patients. We did not observe changes of B cell differentiation subsets in nRA patients. No evidence for B cell repopulation or compensatory hyperproliferation As numbers of circulating B cells normalised in treated rGCA and rPMR patients, we sought to explain how this was achieved. To that end, we first investigated if the circulating B cell pool was perhaps replenished by immature, transitional B cells from the bone marrow. Since transitional B cells were even further decreased in peripheral blood of treated rGCA and rPMR patients (Fig.3A), it seemed highly unlikely that the circulating B cell pool was repopulated by newly-produced B cells from the bone marrow. Next, we assessed whether B cell numbers were replenished through compensatory hyperproliferation of circulating B cells in rGCA and rPMR patients. Therefore, B cells derived from peripheral blood were stained for the proliferation marker Ki-67 before and after treatment (Fig.3B). Before treatment, the proportions of proliferating B cells in nGCA and nPMR patients were similar to HCs (Fig.3B). Corticosteroid treatment, however, led to decreased peripheral B cell proliferation in rGCA patients and slightly tended to reduce proliferation in rPMR patients. As B cell numbers did not seem to be replenished via compensatory hyperproliferation or enhanced bone marrow output, it seemed unlikely that B cells are actually lost in nGCA and nPMR. To further preclude a prominent role of B cell apoptosis in both diseases, we assessed circulating B cells for expression of the anti-apoptotic regulator Bcl-2. B cells of GCA and PMR patients indeed demonstrated a normal per-cell expression level of Bcl-2 during active disease and remission (Fig.3C). Next, we further investigated the composition of the peripheral B cell pool in treated rGCA and rPMR patients. In particular mature-naive B cells and memory B cells seemed to return to the circulation of treated rGCA and rPMR patients. Numbers of mature-naive B cells, IgM only memory B cells and switched memory B cells normalized in rGCA and rPMR 8 John Wiley & Sons

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patients (data not shown). Whereas circulating numbers of unswitched memory B cells remained normal in rPMR patients, we observed a further expansion of unswitched memory B cells in treated rGCA patients (Fig.3D). In contrast, the circulating number of double-negative B cells tended to remain decreased in rPMR patients, and the same was observed for plasmablasts in both rGCA and rPMR patients (data not shown). Taken together, both maturenaive and memory B cell subsets returned to the circulation in GCA and PMR patients in remission. Serum levels of BAFF are increased in newly-diagnosed GCA and PMR patients As our results did not provide evidence for enhanced B cell apoptosis during active disease or compensatory production of B cells during remission, we hypothesized that the mature-naive and memory B cells had been either marginalized or redistributed during active GCA and PMR. To test this, the B cell growth factor BAFF was used as an indirect marker for the presence or absence of B cells in the circulation. Serum levels of BAFF highly depend on the number of circulating B cells and typically rise when B cells are absent from the circulation (30, 31). Indeed, serum BAFF levels were increased in untreated nGCA and nPMR patients (Fig.4A). Following treatment, serum BAFF levels decreased in rGCA and rPMR patients (Fig.4B), but rose again during relapse (Fig.4A). Serum BAFF levels inversely correlated with circulating B cell numbers in GCA and PMR patients (Spearman’s rho = -0.43; p = 0.0015). Overall, our data indicated that B cells were redistributed during active GCA and PMR, and returned during remission. Only non-physiological BAFF levels induce IL-6 in monocytes Although serum BAFF levels are primarily influenced by circulating B cell numbers (30), soluble BAFF can also activate monocytes to produce IL-6 (32, 33). Therefore, we hypothesized that low B cell counts in GCA and PMR patients may indirectly contribute to production of IL-6 in monocytes via elevated serum BAFF levels. In accordance with this hypothesis, we found a strong positive correlation between serum levels of BAFF and IL-6 in 9 John Wiley & Sons


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GCA and PMR patients (Fig.4C). However, non-physiological high BAFF concentrations were required for significant in vitro production of IL-6 in monocytes of HCs and rGCA/rPMR patients. Furthermore, monocytes of rGCA/rPMR seemed to require twice as high concentrations of soluble BAFF for induction of IL-6 in comparison to HCs. In our assays, we excluded indirect stimulation of monocytes via other BAFF-induced cytokines, as cytokine excretion was blocked by Brefeldin A. Soluble BAFF did not induce IL-6 production in B cells or T cells (data not shown). Although we did not study monocytes of nGCA/nPMR patients, our data indicate that significant IL-6 production by monocytes can only occur when these cells are exposed to non-physiological concentrations of BAFF. Therefore, it is unlikely that BAFF-induced activation of peripheral monocytes explains the elevated IL-6 levels in GCA and PMR patients. Beffector cells of GCA and PMR patients show increased potential to produce IL-6 Since B cells can modulate autoimmune responses by producing cytokines (34), we analyzed whether circulating TNF-α+ B cells (Beffector cells) or IL-10+ B cells (Bregulatory cells) were redistributed in nGCA and nPMR patients (Fig.5A). Circulating Beffector cells (TNF-α+IL-10-) were decreased in nGCA and nPMR patients, whereas circulating numbers of Bregulatory cells (TNF-α-IL-10+) were normal (Fig.5B). We also observed a small fraction of TNF-α+IL-10+ B cells, but their numbers were similar in nGCA and nPMR patients and HCs (data not shown). Additional phenotypical analysis of Beffector and Bregulatory cells showed that production of TNFα and IL-10 varied among B cell differentiation subsets. Whereas Beffector cells were found among all memory B cells subsets, Bregulatory cells resided in the unswitched memory B cell population only (Suppl. Fig. 1). Since Beffector cells may produce the pro-inflammatory cytokine IL-6 (34), we analyzed the returning Beffector cells in rGCA and rPMR patients for production of IL-6 (Fig.5C). As expected, TNF-α+ Beffector cells produced more IL-6 than TNF-α- B cells (Fig.5D).

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Interestingly, the percentage of IL-6 producing cells was significantly expanded among Beffector cells of rGCA and rPMR patients. We also observed a slight increase in IL-6 producing cells among TNF-α- B cells of rGCA and rPMR patients. The enhanced potential to produce IL-6 was also noted when rGCA and rPMR patients were analyzed separately (data not shown). B cells in temporal arteries of GCA patients To determine whether B cells can be detected in inflamed arteries of GCA patients, we performed immunohistochemistry for CD20+ B cells on temporal artery biopsies (TABs) of GCA patients (Fig.6A). Only small to moderate numbers of CD20+ B cells were found in the adventitia of all inflamed TABs studied, but rarely in the media and intima (Fig.6B). CD20+ B cells were mostly confined to areas that were also infiltrated by CD3+ T cells. In some TABs, small to moderate numbers of CD138+ plasma cells were detected, primarily in the adventitia. Although B cells were readily detected in TABs from GCA patients, both CD20+ B cells and CD138+ plasma cells were outnumbered by CD3+ T cells.

Discussion Our findings indicate that the distribution of B cells is highly disturbed in newly-diagnosed GCA and PMR patients. A longitudinal analysis of B cells and serum BAFF levels suggested that circulating B cells were redistributed during active GCA and PMR, but quickly returned during remission. More specifically, Beffector cells rather than Bregulatory cells were redistributed during active disease. Beffector cells that returned during remission in GCA and PMR patients, demonstrated an enhanced potential to produce the pro-inflammatory cytokine IL-6. Both newly-diagnosed GCA and PMR patients showed a marked decrease of circulating B cells before corticosteroid treatment. It is well known that GCA and PMR frequently overlap in individual patients (2) and similarities in auto-antibody production by GCA and PMR patients have been reported (12, 14). Our findings further underline the close relationship between GCA and PMR. The decreased B cell numbers seemed characteristic for 11 John Wiley & Sons


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both diseases, whereas we found normal B cell numbers in newly-diagnosed, untreated RA patients. In addition, normal or even increased total B cell numbers have been found in ANCA-associated vasculitis and in other systemic inflammatory diseases (19, 20, 27, 35, 36). Disturbances in B cell homeostasis were also noted in Takayasu arteritis, although absolute numbers of B cells were normal (20). A direct comparison, however, with our data from untreated, newly-diagnosed GCA and PMR patients remains difficult, because the patients with Takayasu arteritis had long-standing disease and had been treated with immunosuppressive drugs (20). The decrease of circulating B cells in newly-diagnosed GCA and PMR patients could not be explained by a loss of B cells. Expression of anti-apoptotic Bcl-2 suggested that circulating B cells were not more susceptible to apoptosis during active disease. Moreover, to correct for an actual loss of B cells (e.g. due to apoptosis), the circulating B cell pool would have to be replenished by newly-produced B cells from the bone marrow, or by hyperproliferation of already existing B cells. We provide evidence against both these mechanisms. Firstly, bone marrow production of new B cells was unlikely, as transitional B cells were nearly ablated by corticosteroid treatment. Instead, mature-naive and memory B cell subsets appeared in the circulation of treated GCA and PMR patients in remission. Although we cannot fully exclude that some transitional B cells are released from the bone marrow early after initiation of treatment, our findings are in accordance with previous studies indicating that corticosteroids induce apoptosis of precursor B cells (37, 38). Secondly, circulating B cells are usually in a relatively quiescent state and have a low proliferation rate in healthy individuals (39). Our data on Ki-67 expression in circulating B cells suggest that this is also true for circulating B cells in GCA and PMR patients before and during treatment. Moreover, proliferation of circulating B cells was even decreased in treated GCA patients, which is a known effect of corticosteroids (40). Thus, we neither found evidence for B cell replenishment 12 John Wiley & Sons

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from the bone marrow, nor for compensatory hyperproliferation of circulating B cells. Taken together, it is highly unlikely that the decrease of circulating B cells in newly-diagnosed GCA and PMR patients resulted from an actual loss of B cells. Instead, our data pointed at redistribution or intravascular marginalization of B cells during active disease. Modulated serum levels of BAFF suggested that B cells were redistributed rather than marginalized in newly-diagnosed GCA and PMR patients. A recent study indicates that the serum level of BAFF in humans is highly dependent on the number of circulating B cells (30). This observation is further supported by findings from trials with B cell depletion therapy (31, 41). Intravascular marginalized B cells would probably still consume BAFF, as they remain in contact with the blood. Interestingly, serum BAFF levels were initially increased in newlydiagnosed GCA and PMR patients, but readily dropped upon the return of B cells during remission. Overall, serum BAFF levels showed a strong inverse correlation with circulating B cell numbers in GCA and PMR patients. Although we cannot exclude that corticosteroids directly suppressed BAFF production to some extent (42), the increased serum levels of BAFF in corticosteroid-treated, relapsing patients would argue against such an effect. High levels of BAFF may favour selection of autoreactive B cells in auto-immune diseases (43). Therefore, it would be interesting to study whether BAFF plays a role in the development of the various auto-antibodies reported in GCA and PMR (11-15). Although BAFF may also induce IL-6 production by monocytes (32, 33), we here demonstrate that this process requires nonphysiological BAFF concentrations that were much higher than the BAFF levels measured in the serum of newly-diagnosed GCA and PMR patients. Of note, the return of B cells towards the circulation in treated rGCA and rPMR patients was not an effect of corticosteroid treatment per se. Although corticosteroids can influence the homing of B cells in healthy humans, they usually direct B cells towards tissues instead of the circulation (38). Furthermore, B cells only returned in treated patients that were 13 John Wiley & Sons


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in remission, but not in treated patients suffering from relapse. Thus, the redistribution and return of B cells towards the circulation seemed to be associated with disease activity rather than corticosteroid treatment. Although lymphocytes can be redistributed towards peripheral sites of inflammation, inflamed arteries and joints are likely not the most important homing site for B cells during active GCA and PMR, respectively. In accordance with previous studies (6-10), we observed small to moderate numbers of B cells in temporal arteries of GCA patients. As B cells were not the predominant cells in the inflamed arteries, it seems unlikely that the decrease of circulating B cells in active GCA patients was solely the effect of B cell migration towards inflamed arteries. Moreover, circulating numbers of B cells did not correlate with the number of inflamed arteries on the FDG-PET/CT scan in GCA patients with large vessel involvement. In addition, others have shown that very few B cells are present in inflamed synovial tissue of PMR patients (5). Therefore, we conclude that peripheral sites of inflammation, i.e. arteries and joints, are unlikely the primary homing sites for B cells during active GCA and PMR. Although the exact reservoir for B cells during active GCA and PMR remains to be established, secondary lymphoid tissues likely represent important homing sites for redistributed B cells in both diseases. Here, activated B cells can undergo isotype-switching under the influence of T cell help (44). Indeed several findings indicate that enhanced B cell activation and isotype-switching occur in GCA and PMR patients. The prevalence of various IgG auto-antibodies is increased in both GCA and PMR patients (11-14). In GCA patients, we also noted a profound expansion of circulating unswitched memory B cells. This finding may suggest that B cells in secondary lymphoid tissues of GCA patients are also activated in the absence of T cell help (45). Interestingly, unswitched memory B cells are induced/maintained by signalling of Toll-like receptors that are also implicated in the pathogenesis of GCA, such as Toll-like receptor-4 (46). 14 John Wiley & Sons

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Ample evidence indicates that B cells not only produce antibodies, but also cytokines that modulate immune responses (34). Intriguingly, inflammatory Beffector cells rather than suppressive Bregulatory cells seemed to be redistributed in newly-diagnosed GCA and PMR patients. . Recently, decreased numbers of IL-10 producing Bregulatory cells were found in ANCA-associated vasculitis (47), whereas normal numbers of Bregulatory cells were found in other auto-immune diseases (22). Our findings indicated that circulating Bregulatory cells may not be subject to redistribution in newly-diagnosed GCA and PMR patients. Only TNF-α producing Beffector cells (34) seemed to leave the circulation during active disease. Interestingly, returning Beffector cells of GCA and PMR patients demonstrated an enhanced potential to produce IL-6. This finding is relevant, as IL-6 producing B cells may potentiate T cell-mediated autoimmunity in humans (24). More specifically, IL-6 is currently thought to promote the pathogenic Th17 response in GCA and PMR (4, 48). As IL-6 production by B cells is promoted by T cell help (24), and IL-6 is important for the development of Th17 cells (4), our findings point towards a delicate interplay between B cells and T cells in GCA and PMR patients. It would be interesting to evaluate whether IL-6R blockade or B cell depletion therapy can ameliorate disease activity in GCA and PMR patients. In conclusion, we demonstrate for the first time that the homeostasis of B cells is highly disturbed in newly-diagnosed GCA and PMR patients. Circulating B cells, including Beffector cells, are decreased in the circulation during active disease, but return during corticosteroid-induced remission. B cells likely contribute to the enhanced IL-6 response of GCA and PMR patients. Our results justify further studies into B cells as biomarkers and possible targets for treatment in GCA and PMR.



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Acknowledgements We thank Anna Richt Miedema for examining the healthy volunteers participating in the study and Pedro Gabriel Lorencetti for his technical assistance. We thank Suzanne Arends for her statistical advice. We thank Riemer Slart and Menno Stellingwerf for evaluating the FDGPET/CT scans. We are grateful to all study participants who kindly donated blood for this study. This work was supported by grants from MSD and from the Dutch Arthritis Association (NC Smit Fonds, Reumafonds grant no. 9-1-201).

Author contributions All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. K. van der Geest, E. Brouwer and A. Boots had full access to all the data in the study and take full responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Van der Geest, Abdulahad, Brouwer, Boots. Acquisition of data. Van der Geest, Chalan, Rutgers, Horst, Huitema, Roffel, Roozendaal, Kluin, Brouwer. Analysis and interpretation of data. Van der Geest, Abdulahad, Chalan, Rutgers, Kluin, Bos, Brouwer, Boots.


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25. Hunder GG, Bloch DA, Michel BA, Stevens MB, Arend WP, Calabrese LH, et al. The American College of Rheumatology 1990 criteria for the classification of giant cell arteritis. Arthritis Rheum 1990; Aug;33(8):1122-8.

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31. Pollard RP, Abdulahad WH, Vissink A, Hamza N, Burgerhof JG, Meijer JM, et al. Serum levels of BAFF, but not APRIL, are increased after rituximab treatment in patients with primary Sjogren's syndrome: data from a placebo-controlled clinical trial. Ann Rheum Dis 2013; Jan;72(1):146-8.

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Figure legends Figure 1. Absolute B cell numbers in GCA and PMR patients before and after treatment. (A) Absolute numbers of peripheral blood CD19+ B cells in healthy controls (HCs; n=40) versus corticosteroid/DMARD-free, newly-diagnosed GCA patients (nGCA; n=16), PMR patients (nPMR; n=18) and RA patients (nRA; n=11). In addition, B cell numbers are shown for 4 relapsing GCA and 4 relapsing PMR patients combined (RLPS GCA/PMR). These relapses occurred after 3 months of treatment. (B) Absolute numbers of circulating CD19+ B cells in newly-diagnosed GCA patients (nGCA), and after 2 weeks (n=11) and 3 months (n=12) of corticosteroid treatment. (C) Absolute numbers of circulating CD19+ B cells in newly-diagnosed PMR patients (nPMR), and after 2 weeks (n=10) and 3 months (n=11) of corticosteroid treatment. (D) Inverse correlation between absolute B cell numbers in peripheral blood of GCA and PMR patients and ESR, as determined by Spearman’s rank correlation coefficient. Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 2. B cell differentiation subsets in newly-diagnosed GCA and PMR patients. (A) Flow cytometry gating strategy to distinguish differentiation subsets within peripheral blood CD19+ B cells. (B) Representative flow cytometry dot plots for CD27 and IgD staining in CD19+ B cells of a healthy control (HC) and an untreated, newly-diagnosed GCA patient are shown. (C) Absolute numbers of B cell differentiation subsets in healthy controls (HCs; n=26) and corticosteroid/DMARD-free, newly-diagnosed GCA patients (nGCA; n=16), PMR patients (nPMR; n=18) and RA patients (nRA; n=11). In addition, B cell numbers are shown for 4 relapsing GCA and 4 relapsing PMR patients combined (RLPS GCA/PMR). Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.


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Figure 3. B cell differentiation subsets in treated GCA and PMR patients in remission. (A) Representative flow cytometry staining of CD24highCD38high transitional B cells in CD19+CD27-IgD+ B cells of a GCA patient before and after 3 months of corticosteroid treatment. Furthermore, absolute numbers are shown for transitional B cells in healthy controls (HCs; n=26), newly-diagnosed GCA patients (nGCA; n=16), GCA patients in remission after 3 months of corticosteroid treatment (rGCA; n=12), newly-diagnosed PMR patients (nPMR; n=18) and PMR patients in remission after 3 months of corticosteroid treatment (rPMR; n=11). (B) Representative flow cytometry staining of Ki-67 in CD19+ B cells of a GCA patient before and after 3 months of corticosteroid treatment. Proportions of Ki-67+ cells within CD19+ B cells are shown for HCs (n=14), nGCA (n=13), rGCA (n=8), nPMR (n=12) and rPMR (n=7) patients. (C) Mean fluorescence intensity (MFI) for Bcl-2 in CD19+ B cells of HCs (n=8), nGCA (n=4), rGCA (n=4), nPMR (n=4) and rPMR (n=4) patients. (D) Absolute numbers of unswitched memory B cells in HCs (n=26), nGCA (n=16), rGCA (n=12), nPMR (n=18) and rPMR (n=11) patients. Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 4. Serum BAFF levels in GCA and PMR patients before and after treatment. (A) Serum levels of BAFF in healthy controls (HCs; n=16) and in untreated, newly-diagnosed GCA (nGCA; n=14) and PMR (nPMR; n=13) patients and in 4 relapsing GCA and 3 relapsing PMR patients combined (RLPS GCA/PMR). (B) Serum levels of BAFF in HCs, nGCA patients and treated GCA patients in remission (rGCA; n=11); and in HCs, nPMR patients and treated PMR patients in remission (rPMR; n=11). (C) Positive correlation between serum BAFF levels and serum IL-6 levels in peripheral blood of GCA and PMR patients, as determined by Spearman’s rank correlation coefficient. (D) Representative flow cytometry



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staining of intracellular IL-6 in unstimulated CD14+ monocytes (medium + brefeldin A) and BAFF stimulated CD14+ monocytes (2000 ng/mL soluble recombinant BAFF + brefeldin A) of a GCA patient in remission. Median percentages of IL-6+ CD14+ monocytes from HCs (n=4), rGCA (n=4) and rPMR (n=4) patients following 4 hour in vitro stimulation with different concentrations of soluble recombinant BAFF. Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 5. Beffector and Bregulatory cells in GCA and PMR patients before and after treatment.

(A) Representative flow cytometry dot plots for intracellular TNF-α and IL-10 staining in peripheral blood CD19+CD22+ B cells of a HC, a newly-diagnosed GCA patient and the same patient in corticosteroid-induced remission. Whole blood samples were stimulated with PMA and Ca2+ ionophore in the presence of brefeldin A during 4 hours. (B) Absolute numbers of TNF+IL-10- Beffector cells and TNF-IL-10+ Bregulatory cells in healthy controls (HCs; n=36), newly-diagnosed GCA patients (nGCA; n=8), treated GCA patients in remission (rGCA; n=7), newly-diagnosed PMR patients (nPMR; n=7) and treated PMR patients in remission (rPMR; n=6). (C) Representative flow cytometry dot plots for intracellular TNF-α and IL-6 staining in peripheral blood CD19+CD22+ B cells of a HC and rGCA patient. Cells were stimulated as mentioned above. (D) Percentage of TNF-α+ Beffector cells and TNF-α- B cells producing IL-6 in HCs (n=12) and GCA/PMR patients (n=11). Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.


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Figure 6. B cells in temporal artery biopsies of GCA patients.

(A) Immunohistochemistry staining for CD20+ B cells, CD138+ plasma cells and CD3+ T cells in a representative inflamed temporal artery biopsy of a GCA patient. (B) Semiquantitative scoring for B cells, plasma cells and T cells in inflamed temporal artery biopsies of 6 GCA patients. Semi-quantitatively scoring was performed by two independent investigators on a five-point scale (0-4): 0 = no positive cells, 1 = occasional positive cells, 2 = moderate number of positive cells, 3 = large number of positive cells, 4 = very large number of positive cells. The scores of both investigators were averaged. Bars represent mean and SEM.

Supplemental table 1. Patient characteristics. Characteristics of newly-diagnosed, corticosteroid/DMARD-free GCA patients (nGCA), PMR patients (nPMR) and RA patients (nRA) and healthy controls (HCs). No. = number. LVV = large vessel vasculitis. TAB = temporal artery biopsy. ESR = erythrocyte sedimentation rate. CRP = C-reactive protein. SEM = standard error of the mean. NA = not applicable. Supplemental figure 1. TNF-α and IL-10 production in B cell differentiation subsets. Whole blood samples of rGCA (n=5) and rPMR (n=5) patients were stimulation with PMA and Ca2+ ionophore in the presence of brefeldin A for 4 hours. Data are shown for GCA and PMR patients combined. (A) Intracellular TNF-α and IL-10 was measured in four distinct CD27/IgD defined B cell differentiation subsets. (B) Pie charts represent median proportions of TNF-α+IL-10-, TNF-α+IL-10+, TNF-α-IL-10+ and TNF-α-IL-10- cells among the four CD27/IgD defined B cell differentiation subsets.



Figure 1. Absolute B cell numbers in GCA and PMR patients before and after treatment. (A) Absolute numbers of peripheral blood CD19+ B cells in healthy controls (HCs; n=40) versus corticosteroid/DMARD-free, newly-diagnosed GCA patients (nGCA; n=16), PMR patients (nPMR; n=18) and RA patients (nRA; n=11). In addition, B cell numbers are shown for 4 relapsing GCA and 4 relapsing PMR patients combined (RLPS GCA/PMR). These relapses occurred after 3 months of treatment. (B) Absolute numbers of circulating CD19+ B cells in newly-diagnosed GCA patients (nGCA), and after 2 weeks (n=11) and 3 months (n=12) of corticosteroid treatment. (C) Absolute numbers of circulating CD19+ B cells in newly-diagnosed PMR patients (nPMR), and after 2 weeks (n=10) and 3 months (n=11) of corticosteroid treatment. (D) Positive correlation between absolute B cell numbers in peripheral blood of GCA and PMR patients and ESR, as determined by Spearman’s rank correlation coefficient. Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

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69x211mm (300 x 300 DPI)

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Figure 2. B cell differentiation subsets in newly-diagnosed GCA patients. (A) Flow cytometry gating strategy to distinguish differentiation subsets within peripheral blood CD19+ B cells. (B) Representative flow cytometry dot plots for CD27 and IgD staining in CD19+ B cells of a healthy control (HC) and an untreated, newly-diagnosed GCA patient are shown. (C) Absolute numbers of B cell differentiation subsets in healthy controls (HCs; n=26) and corticosteroid/DMARD-free, newly-diagnosed GCA patients (nGCA; n=16), PMR patients (nPMR; n=18) and RA patients (nRA; n=11). In addition, B cell numbers are shown for 4 relapsing GCA and 4 relapsing PMR patients combined (RLPS GCA/PMR). Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 177x109mm (300 x 300 DPI)

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Figure 3. B cell differentiation subsets in treated GCA patients in remission. (A) Representative flow cytometry staining of CD24highCD38high transitional B cells in CD19+CD27-IgD+ B cells of a GCA patient before and after 3 months of corticosteroid treatment. Furthermore, absolute numbers are shown for transitional B cells in healthy controls (HCs; n=26), newly-diagnosed GCA patients (nGCA; n=16), GCA patients in remission after 3 months of corticosteroid treatment (rGCA; n=12), newlydiagnosed PMR patients (nPMR; n=18) and PMR patients in remission after 3 months of corticosteroid treatment (rPMR; n=11). (B) Representative flow cytometry staining of Ki-67 in CD19+ B cells of a GCA patient before and after 3 months of corticosteroid treatment. Proportions of Ki-67+ cells within CD19+ B cells are shown for HCs (n=14), nGCA (n=13), rGCA (n=8), nPMR (n=12) and rPMR (n=7) patients. (C) Mean fluorescence intensity (MFI) for Bcl-2 in CD19+ B cells of HCs (n=8), nGCA (n=4), rGCA (n=4), nPMR (n=4) and rPMR (n=4) patients. (D) Absolute numbers of unswitched memory B cells in HCs (n=26), nGCA (n=16), rGCA (n=12), nPMR (n=18) and rPMR (n=11) patients. Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 176x100mm (300 x 300 DPI)

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Figure 4. Serum BAFF levels in GCA patients before and after treatment. (A) Serum levels of BAFF in healthy controls (HCs; n=16) and in untreated, newly-diagnosed GCA (nGCA; n=14) and PMR (nPMR; n=13) patients and in 4 relapsing GCA and 3 relapsing PMR patients combined (RLPS GCA/PMR). (B) Serum levels of BAFF in HCs, nGCA patients and treated GCA patients in remission (rGCA; n=11); and in HCs, nPMR patients and treated PMR patients in remission (rPMR; n=11). (C) Positive correlation between serum BAFF levels and serum IL-6 levels in peripheral blood of GCA and PMR patients, as determined by Spearman’s rank correlation coefficient. (D) Representative flow cytometry staining of intracellular IL-6 in unstimulated CD14+ monocytes (medium + brefeldin A) and BAFF stimulated CD14+ monocytes (2000 ng/mL soluble recombinant BAFF + brefeldin A) of a GCA patient in remission. Median percentages of IL-6+ CD14+ monocytes from HCs (n=4), rGCA (n=4) and rPMR (n=4) patients following 4 hour in vitro stimulation with different concentrations of soluble recombinant BAFF. Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 172x97mm (300 x 300 DPI)

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Figure 5. Beffector and Breg cells in GCA before and after treatment. (A) Representative flow cytometry dot plots for intracellular TNF-α and IL-10 staining in peripheral blood CD19+CD22+ B cells of a HC, a newly-diagnosed GCA patient and the same patient in corticosteroidinduced remission. Whole blood samples were stimulated with 40nM PMA and Ca2+ ionophore in the presence of brefeldin A during 4 hours. (B) Absolute numbers of TNF+IL-10- Beffector cells and TNF-IL-10+ Bregulatory cells in healthy controls (HCs; n=36), newly-diagnosed GCA patients (nGCA; n=8), treated GCA patients in remission (rGCA; n=7), newly-diagnosed PMR patients (nPMR; n=7) and treated PMR patients in remission (rPMR; n=6). (C) ) Representative flow cytometry dot plots for intracellular TNF-α and IL-6 staining in peripheral blood CD19+CD22+ B cells of a HC and rGCA patient. Cells were stimulated as mentioned above. (D) Percentage of TNF-α+ Beffector cells and TNF-α- B cells producing IL-6 in HCs (n=12) and GCA/PMR patients (n=11). Bars represent mean and SEM. Statistical significance of patients versus HCs was tested by Mann Whitney U test and is indicated as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 179x95mm (300 x 300 DPI)

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Figure 6. B cells in temporal artery biopsies (A) Immunohistochemical staining for CD20+ B cells, CD138+ plasma cells and CD3+ T cells in a representative inflamed temporal artery biopsy of a GCA patient. (B) Semi-quantitative scoring for B cells, plasma cells and T cells in inflamed temporal artery biopsies of 6 GCA patients. Semi-quantitatively scoring was performed by two independent investigators on a five-point scale (0-4): 0 = no positive cells, 1 = occasional positive cells, 2 = moderate number of positive cells, 3 = large number of positive cells, 4 = very large number of positive cells. The scores of both investigators were averaged. Bars represent mean and SEM. 156x102mm (300 x 300 DPI)

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giant cell arteritis.

Polymyalgia rheumatica and giant-cell arteritis in a black woman.

Prognosis of polymyalgia rheumatica and giant cell arteritis.

Clinical practice. Giant-cell arteritis and polymyalgia rheumatica.

Polymyalgia rheumatica and giant cell arteritis: a seven-year survey.

giant cell arteritis.

Relation of giant cell arteritis to polymyalgia rheumatica.

CT.

Serum markers associated with disease activity in giant cell arteritis and polymyalgia rheumatica.

Epidemiologic and immunogenetic aspects of polymyalgia rheumatica and giant cell arteritis in northern Italy.

Microheterogeneity of alpha 1-antichymotrypsin in the management of giant-cell arteritis and polymyalgia rheumatica.

Hypothyroidism in polymyalgia rheumatica and giant cell arteritis: lack of any association.

Autoimmune thyroid disease and the polymyalgia rheumatica-giant cell arteritis syndrome.

The prevalence of giant cell arteritis and polymyalgia rheumatica in a UK primary care population.

Development of giant cell (temporal) arteritis in a patient 'adequately' treated for polymyalgia rheumatica.

Associations between polymyalgia rheumatica and giant cell arteritis and 12 cardiovascular diseases.

Association of polymyalgia rheumatica and giant-cell arteritis with HLA-B8.

Death from unsuspected diverticular disease in patients taking corticosteroids for polymyalgia rheumatica or giant cell arteritis.

Anticardiolipin antibodies in polymyalgia rheumatica-giant cell arteritis: association with severe vascular complications.

Giant cell arteritis and polymyalgia rheumatica after reexposure to a statin: a case report.

FDG positron emission tomography of giant cell arteritis with polymyalgia rheumatica.

Polymyalgia rheumatica and temporal arteritis.

Risk of malignancy in patients with giant cell arteritis and polymyalgia rheumatica: a systematic review and meta-analysis.

Level of Adherence to Prophylactic Osteoporosis Medication amongst Patients with Polymyalgia Rheumatica and Giant Cell Arteritis: A Cross-Sectional Study.