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Background Autoantibodies contribute significantly to the pathogenesis of systemic lupus erythematosus (SLE). The long-lived plasma cells (LLPC) secreting such autoantibodies are refractory to conventional immunosuppressive treatments. Although they are generated long before the disease becomes clinically apparent, it is unknown whether their generation continues in the established disease. Objectives Here, we analyze the generation of autoreactive LLPCs in lupus-prone NZB/W F1 mice over their lifetime, and LLPC regeneration after depletion. Methods BrdU pulse-chase experiments were used to follow the establishment of the LLPS compartment. Bortezomib and cyclophosphamide therapy was used to deplete the LLPCs. Results Autoreactive LLPCs are established in the spleen and bone marrow of lupus-prone mice very early in ontogeny, before week 8 and before the onset of symptoms. The generation of LLPCs then continues throughout life. LLPC counts in the spleen plateaued by week 10, but continued to increase in the bone marrow. When LLPCs are depleted by the proteasome inhibitor bortezomib, their numbers regenerate within two weeks. Persistent depletion of LLPCs was achieved only by combining a shot of bortezomib with maintenance therapy, e.g., cyclophosphamide, depleting the precursors of LLPCs or preventing their differentiation into LLPCs. Conclusions In lupus-prone NZB/W F1 mice, autoreactive LLPCs are generated throughout life. Their sustained therapeutic elimination requires both the depletion of LLPCs and the inhibition of their regeneration. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.3408

Objectives The proteasome inhibitor bortezomib, approved for the therapy of multiple myeloma, depletes plasma cells (PCs) and ameliorates nephritis in mouse models of systemic lupus erythematosus (SLE). Here, we analyzed the efficacy of bortezomib as induction therapy in patients with refractory SLE. Methods Twelve patients with active SLE were included in this prospective multicentre cohort study. The patients received one to four cycles of intravenous bortezomib (Velcade®) 1.3mg/m 2 as “off-label” treatment. Disease activity was evaluated using the SELENA-SLEDAI score. We determined serum concentrations of anti–double-stranded DNA (anti-dsDNA) and protective antibodies. Multicolor flow cytometry was performed to analyze peripheral blood B and PC subsets as well as Siglec-1 expression on monocytes as surrogate marker for type I interferon (IFN) activity. Results The disease activity significantly decreased upon induction therapy with bortezomib and remained stable for the following 3 months under maintenance therapies. Treatment-related adverse events were mild or moderate. During proteasome inhibition, serum antibody concentrations significantly declined with greater reductions in anti-dsDNA (∼60%) than protective (∼30%) antibodies. Upon bortezomib treatment, numbers of HLA-DR + short-lived (p=0.024) and HLA-DR – long-lived (p=0.038) peripheral blood PCs were strongly decreased, whereas circulating B cells remained virtually unaffected. Notably, PC numbers significantly increased in-between cycles. Siglec-1 expression on monocytes significantly declined (p Conclusions Bortezomib targeting PCs and type I IFN activation may represent an effective treatment option with rather low toxicity in refractory SLE patients. Bortezomib efficiently induces short-term remissions but requires maintenance treatment inhibiting PC regeneration for sustained efficacy. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5884

Background and Objectives Long-lived plasma cells (LLPC) contribute significantly to the production of pathogenic autoantibodies in Systemic Lupus Erythematosus (SLE), the prototype of systemic autoimmune diseases. These cells are refractory to conventional immunosuppressive therapies and thus represent an unmet therapeutic challenge. In the current view, LLPC are generated early in ontogeny and no longer formed later in disease pathogenesis, when constant generation of short-lived plasma cells is supposed to be a hallmark of pathology. However, the homeostasis of autoreactive LLPC-compartment has not been characterised so far. Therefore, in this study we rigorously analysed the dynamics of generation, maintenance and replacement of LLPC in NZB/W mice, a murine model of SLE. Materials and Methods NZB/W mice of different ages (4 to 29 week) were fed with bromodeoxyuridine (BrdU) in their drinking water for two weeks in order to discriminate between cells generated during and before the feeding period and to track the cells and their replacement over time. Next, LLPC were depleted in mice with a stable splenic LLPC-compartment by two injection of bortezomib (0.75 mg/kg BW) in combination or not with a dose of 35 mg/kg BW cyclophosphamide every fourth day. The mice were sacrificed at different time points after BrdU feeding or LLPC-depletion and bone marrow and spleen LLPC were enumerated by FACS and ELISPOT. Results Generation of autoreactive LLPC in spleen and bone marrow starts very early in ontogeny before the onset of symptoms. LLPC-generation continues and is maintained throughout life, reaching a plateau in the spleen but showing persistently increasing numbers and replacement in bone marrow. When LLPCs are efficiently depleted by bortezomib, their numbers fully recover within 2 weeks after its withdrawal. After termination of bortezomib, a persistent depletion of LLPC was only maintained if the precursors of the LLPC were continuously targeted e.g. using cyclophosphamide. Conclusion Our results provide relevant insights into the disturbed homeostasis of B cell and LLPC showing that, in autoimmunity, the LLPC-compartment manifest an unforeseen dynamism that is related to B cell hyperactivity. Our data disprove the idea of a “therapeutic window of opportunity” as the generation of LLPC starts very early in ontogeny. Moreover, the continuous generation and replacement of autoreactive-LLPC has an impact on the treatment of systemic autoimmune diseases suggesting that the depletion of autoreactive-LLPC has to be connected with the prevention of regeneration of these cells through the targeting of B cell activation and differentiation.

Background The proteasome inhibitor bortezomib, approved for the treatment of multiple myeloma, has been demonstrated to deplete short- and long-lived plasma cells (PCs) and ameliorate nephritis in murine models of systemic lupus erythematosus (SLE). Here, we aimed to analyse the effect of bortezomib in refractory SLE patients. Methods In this single-centre cohort study, eight SLE patients with active disease despite immunosuppressive treatment received up to four 21-day cycles of bortezomib 1.3mg/m 2 , on days 1, 4, 8, and 11 by intravenous injection as an “off-label” treatment. Multicolor flow cytometry was performed to analyse peripheral blood B and PC subsets as well as Siglec-1 expression on monocytes. Autoantibody and vaccine titres as well as B cell activating factor (BAFF) levels in serum were analysed with ELISA. Results From 2009 until 2012, eight SLE patients received a median of two bortezomib cycles (range one to four). Under proteasome inhibition, disease activity significantly improved with a median SLEDAI reduction from 13 to 4 (p>0.001). Bortezomib treatment had profound effects on antibody titers, with greater reduction in pathogenic (anti-dsDNA-abs. 58.7%) than protective antibody titers (e.g. anti-Tetanus-abs. 29.2%). In addition, total immunoglobulin levels and Siglec-1 expression on monocytes (as surrogate for type I interferon) significantly decreased (p = 0.016), whereas BAFF and complement levels significantly increased. While circulating B-cell numbers remained unaffected, bortezomib treatment resulted in a significant depletion of both HLA-DR + short-lived (55.5% reduction, p = 0.024) and HLA-DR- long-lived (53.5% reduction, p = 0.038) peripheral blood PCs. Also IgA secreting PCs were depleted (61.8% reduction). Nevertheless, PCs were rapidly regenerated with a median increase of 268.8% within 10 days from the last bortezomib application. Conclusion Proteasome inhibition with bortezomib has beneficial effects on disease manifestations in SLE by PC depletion and inhibition of type I interferon but regeneration of PCs is not prevented. Overall, proteasome inhibition has demonstrated the therapeutic relevance of targeting autoreactive memory plasma cells as such, and it constitutes a new therapeutic option. Nevertheless, fur sustained efficacy, proteasome inhibition needs to be combined with therapeutic approaches targeting B cell activation and differentiation to inhibit PC regeneration.

Background Active systemic lupus erythematosus (SLE) is associated with increased levels of peripheral blood antibody-secreting plasmablasts which correlate with autoantibody production and disease activity. Plasmablasts which circulate in healthy individuals at very low levels in steady-state express a mucosal phenotype and secrete IgA, but are conditionally expanded by additional IgG-secreting plasmablasts during systemic immune responses. The dynamics of plasmablasts generated in mucosal IgA vs. systemic IgG responses underlying the expanded plasmablast subset in SLE patients and its implication in SLE immunopathogenesis remain unexplored. Objectives To analyse the blood of SLE patients for the presence and characteristics of IgA+ plasmablasts and to evaluate their potential relation to SLE disease activity. Methods Peripheral blood plasmablasts from 30 SLE patients and 20 healthy controls (HD) were enumerated and analysed by multi-parametric flow cytometry using antibodies directed against CD19, CD27, CD20, and analysed for surface co-expression of IgA, CCR10 and beta7 Integrin. Disease activity was assessed by SLEDAI scores. Results IgA+ plasmablasts were detected in the blood of all controls and at significantly higher numbers in SLE patients (HD, IgA+ 1,2 cells/μl; SLE, IgA+ 3,2 cells/μl; p=0.0017). Patients with high disease activity (SLEDAI ≥6) showed significantly higher numbers of circulating IgA+ plasmablasts when compared with less active patients at SLEDAI In SLE patients, expression of CCR10 and beta7 integrin was associated with IgA expression. However, we found that a unique IgA+/CCR10-/beta7- plasmablast subset disproportionately contributed to the expansion of IgA+ plasmablasts in the blood of SLE patients (HD, 25%; SLE, 38%). In contrast, IgA+ plasmablasts expressing CCR10 but not beta7 Integrin were significantly reduced in SLE patients compared to healthy controls (HD, 48%; SLE, 29%; p=0,0182). Conclusions Our data demonstrate that IgA+ plasmablasts contribute to the expanded plasmablast subset in SLE patients related to disease activity. The data implicate enhanced activation and differentiation of mucosal B cells, although the expansion of a distinct IgA+ plasmablast subset lacking mucosal homing receptors beta7 Integrin and CCR10 indicates abnormalities of mucosal B cell activation in these patients. Disclosure of Interest None Declared

Background In Systemic Lupus Erythematosus (SLE) various compartments of the immune system are altered. Type I interferon (IFN) produced by plasmacytoid dendritic cells seems to play a central role. The expression of Sialic Acid–Binding Ig-like Lectin 1 (Siglec-1) on monocytes is a cytometrically measurable surrogate parameter for type I IFN and has been shown to correlate with disease activity in SLE. Increased B cell activation is also characteristic for active SLE. Frequencies of CD27 ++ plasmablasts in peripheral blood are known to be higher in patients with active disease. Type I IFN can promote T cell dependent and independent B cell activation. Objectives To investigate the combined measurement of Siglec-1 expression and plasmablast frequencies as biomarkers for disease activity in human SLE. Methods After informed consent heparinised blood samples along with clinical and paraclinical data were collected from 12 SLE patients. After red cell lysis FACS staining was performed with fluorescent antibodies against CD14, CD3, CD19, CD20, CD27, CD138 and Siglec-1 plus isotype control. Cells were collected and measured using a BD™ LSR II flow cytometer. Data were analysed using FLOWJO analysis software. For Siglec-1 expression on CD14 + monocytes a ratio was calculated dividing the median fluorescence intensity (MFI) value of Siglec-1 stained by the MFI of isotype control stained cells. Plasmablast frequencies where calculated by gating on mononuclear cells according to forward and sideward scatter, further gating on CD19 + CD3 - cells and than on CD20 - CD27 ++ or CD20 - CD138 + cells, respectively. Results Siglec-1 expression correlated negatively with serum complement (C3) but not with SLEDAI or anti double stranded DNA (dsDNA) antibody titers using the non parametric Spearman test. Both, frequencies of CD20 - CD27 ++ and frequencies of CD20 - CD138 + cells correlated negatively with serum complement and positively with SLEDAI but not with dsDNA antibody titers. This was the case for frequencies in CD19 + B cells as well as in lymphocytes. Furthermore, Siglec-1 expression correlated positively with plasmablast frequencies, again for both, CD20 - CD27 ++ and CD20 - CD138 + cells. Conclusions All measured parameters (Siglec-1 in monocytes, CD20 - CD27 ++ CD19+ cells and CD20 - CD138 + CD19+ cells) seem to be suitable as additional disease activity markers in SLE. The reliability of the single markers and combinations should, of course, be studied in a bigger cohort, and also under various treatment conditions. The correlation of Siglec-1 expression with plasmablast frequencies is a further hint to pathogenetic relations between type I interferon and B cell activation. References Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner M, Backhaus M, Haupl T, Rudwaleit M, Riemekasten G, Radbruch A, Hiepe F, Burmester GR, Grutzkau A. Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum. 2008 Apr;58(4):1136-45. Jacobi AM, Odendahl M, Reiter K, Bruns A, Burmester GR, Radbruch A, Valet G, Lipsky PE, Dorner T. Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus. Arthritis Rheum. 2003 May;48(5):1332-42. Disclosure of Interest None Declared

Background B cells are important players in granulomatosis with polyangiitis (GPA) as suggested by the presence of autoantibodies reacting with specific neutrophil granular enzymes ANCA) in a vast majority of patients as well as the success of B cell depleting therapies in GPA. Renal manifestations in GPA are considered to be directly ANCA – mediated whereas the granulomatous inflammation appears to be mediated by CD4 T cells. Objectives For a better understanding of the possible role of B cells in ANCA-associated vasculitis we analyzed the B cell subsets in the peripheral blood of patients with GPA and found marked changes correlating with disease activity as measured by the BVAS (Birmingham vasculitis activity score) as well as ANCA-levels. Methods 17 patients with GPA (10 with active disease, 7 with inactive disease) were analyzed by flow cytometry as compared to 20 healthy donors. Staining for CD27, CD20, CD19, MHCII, CD3, 4 and 8 was analyzed using flowjo software. Statistical analysis was performed using graph pad prism, and p-values of Results Marked differences (p=0,0018) could be observed in plasma cell counts as well as frequencies in GPA patients (6,5 ±5,06/µl) with a BVAS-score > 0 as compared to those with a BVAS-score =0 (2,5± 1,6/µl) or healthy persons (2,3±1,15/µl). Plasma cell numbers as well as the frequency of plasma cells within all B cells correlated with the BVAS (r= 0,91, p Regarding T cells, there was a significant reduction of CD3 (p=0,01) and CD4 T (p=0,012) cells in patients with active GPA as compared to patients in remission, whereas CD8 T cells did not show any significant changes. Conclusions Plasma cell numbers are increased in patients with active GPA which implies a role for plasma cell mediated effects in active GPA. Presumably, their main role is the production of autoantibodies, but also cytokine production. Independent of their direct contribution to the disease, they may serve as a biomarker of disease activity as they highly correlate with the BVAS. References Hoyer et al, ARD 2012 Disclosure of Interest None Declared

Background Takayasu arteritis and giant cell vasculitis are two forms of large vessel disease mostly affecting women. One of their main differences is the age at onset this being around 20 years for Takayasu’s (TA) and largely above 50 for giant cell vasculitis (GCA). In some patients the affected vessels also differ with a focus on the temporal artery in the aged patients and a stronger focus on the aorta in TA.Until recently T cells were believed to be the major player in those fomrs of large vessel vasculitis. We could show recently that B cell disturbances ca be found in Takayasu arteritis correlating with dsease activity. For giant cell vasculitis data is still lacking about B cell disturbances. Objectives In this study we compare the frequency and absolute number of B cell subsets in the peripheral blood of patients suffering from TA, GCA and Systemic lupus erythematosus (SLE) as compared to healthy controls. Methods Peripheral blood mononuclear cells were analysed ex vivo by flow cytometry according to their expression of CD19, CD20, CD27, IgD and MHCII. Samples from 14 with active GCA, 10 with inactive GCA, 8 with active SLE, 5 with active TA and 25 healthy controls were analysed. Plasma cells were characterized by being CD19 pos, CD27 high, CD20 neg. Switched memory B cells were characterized by being CD19pos, CD27pos IgD neg, Naive B cells were characterized as being CD27 neg IgD pos. Double neg cells were CD19 pos CD27 neg IgD neg. Calculations were done using Graph Pad prism using Mann-Whitney-test and p- values of less than 0.05 were taken as significant. Results Overall B cell numbers are comparable in GCA, TA and healthy donors whereas they were found to be reduced in SLE as was reported previously. Regarding plasma cells frequency we could detect a significant increase in patients with active GCA, active TA and obviously SLE as compared to healthy controls whereas frequency in patients with inactive disease were only slightly increased. Regarding absolut numbers this change was only detecable for patients with active GCA. For MHCII-high plasma cells a reduction as compared to healthy controls could be observed that did not reach significance. In the other B cell subsets a significant reduction could be found between healthy donors and patients with active GCA regarding the naive B cells (frequency and absolute numbers p=0,01 and p=0,005). In Takayasu patients a similar change could be observed but not being significant. For the frequency a significant increase could be observed regarding the switched memory B cells (p=0,003) that was nearly as pronounced in inactive GCA but was not observed in TA and SLE. Conclusions Here we show for the first time that disturbances in B cell subsets in the peripheral blood can be observed not only in TA but also in patients with GCA. The observed changes differ between giant cell vasculitis and TA and are only to some extend comparable to the ones described in SLE. Therefore the flowcytometric analysis of B cell substes in the blood could be a tool to differentiate between thoses diseases and are another hint for a different B-cell-pathomechanism for TA and GCA. In addition B cells might represent an interesting therapeutic target for new drugs. Disclosure of Interest None Declared

Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the generation of pathogenic antibodies directed against a variety of autoantigens. We have previously shown that long-lived plasma cells can contribute to autoimmune pathology by secreting pathogenic autoantibodies. These cells are resistant to conventional immunosuppressive drugs, irradiation and B cell depletion. So far, immunoablation using antithymocyte globulin (ATG) followed by autologous hematopoietic stem cell transplantation and the proteasome inhibitor bortezomib are able to deplete long-lived plasma cells. Objectives This study is aimed to test strategies for selective depletion of autoreactive long-lived plasma cells and to prevent their new generation in NZB/W mice representing a model of SLE. Methods We studied immunoablative abilities of mixture of monoclonal antibodies in young (2 month-old) and old (6 month-old) NZB/W mice. We used double i.p coinjection of 200μg anti-LFA-1 and 200μg anti-VLA-4 in a 2-d interval, three times i.v injection of 0,75mg/kg bortezomib in 36-h intervals to achieve ablation of long-lived plasma cells. Furthermore, as B cells will contribute to the repopulation of plasma cells, we also co-treated mice with intravenous injections of 10mg/kg anti-CD20 and 250μg anti-B220 to deplete those plasma cell precursors. All injections were performed within one week. BrdU-feeding was started one week prior to treatment and continued for the whole treatment period (total 2 weeks) to analyze the therapeutic effect on the long-lived plasma cell compartment. The effect of this therapeutic regimen on B cells and plasma cells was analyzed 12 hour, 3 and 7 days after the last injection of Bortezomib by flow cytometry. Results Flowcytometric analysis at hour 12 shows that the mixture of anti-CD20 and anti-B220 (targeting B cells), anti-VLA-4, anti-LFA-1 and bortezomib (targeting Plasma cells) was able to deplete all subsets of B cells, short-lived and long-lived plasma cells in spleen and bone barrow of young and old NZB/W mice. However FACS analysis on days 3 and 7 indicates a repopulation process by the graduate increase of short-lived and long-lived plasma cells. Long-lived plasma cells reached up to 7% and 41% of controls on day 7 in spleen and bone marrow respectively. These Results demonstrate the fast kinetics of plasma cell recovery after plasma cell depletion in the lupus-prone NZB/W mouse model. Conclusions The combination treatment with the targeting of adhesion molecules and the use of the proteasome inhibitor (bortezomib) can deplete almost all long-lived plasma cells in bone marrow and spleen but their repopulation is fast. Therefore we will have to to find better ways to target the plasma cell precursors for longlasting depletion. References Neubert, K., et al., The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis. Nat Med, 2008. 14(7): p. 748-55 DiLillo, D.J., et al., Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice. J Immunol, 2008. 180(1): p. 361-71. Bekar, K.W., et al., Prolonged effects of short-term anti-CD20 B cell depletion therapy in murine systemic lupus erythematosus. Arthritis Rheum, 2010. 62(8): p. 2443-57. Disclosure of Interest None Declared

Backgroundand objectives Long-lived memory plasma cells secreting pathogenic autoantibodies are resistant to conventional immunosuppression and B cell targeting therapies. Bortezomib, an approved drug for relapsing multiple myeloma, efficiently depletes short- and long-lived plasma cells and ameliorates nephritis in murine lupus models. Therefore, the authors studied the efficacy of bortezomib in refractory SLE patients in a case series. Materials and methods At three university centers, 13 SLE patients refractory or intolerant to cyclophosphamide, MMF and/or rituximab were treated with bortezomib intravenously at a dose of 1.3 mg/m2 body surface area on days 1, 4, and 8 and, in some cases, on day 11. Most patients received 20mg of dexamethasone together with bortezomib. Treatment cycles were repeated up to four times with an interruption of usually 10 to 14 days between cycles. The following clinical and laboratory parameters were monitored: SLEDAI disease activity score, 24 h proteinuria, creatinine clearance, circulating plasma cells, complement C3 and C4, IgG, IgA, IgM, antibodies to dsDNA and ENA, and vaccine titers. Results SLEDAI and antibody levels significantly decreased under bortezomib while complement levels increased. In seven patients with active nephritis, proteinuria declined within 6 weeks of treatment, and normalised after four months in one case. Anti-dsDNA antibodies decreased by up to 90%, anti-ENA and protective vaccine titers by up to 50%, and immunoglobulin levels by up to 30%. Circulating plasmablasts dropped substantially. Serious side effects were not observed. One patient experienced myalgia, fever and headache 1 day after the first 3 bortezomib doses. Three of five patients treated with four bortezomib injections per cycle developed polyneuropathies, which were reversible upon discontinuation of treatment. One patient developed reversible thrombocytopenia after four treatment cycles. Conclusions The proteasome inhibitor bortezomib may effectively reduce disease activity in refractory SLE patients by depleting plasma cells. The data encourage initiation of clinical trials with bortezomib in refractory SLE and support the relevance of plasma cells in the pathogenesis of this autoimmune disease.

Background and objectives Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease. Compelling evidence suggests that autoreactive memory plasma cells (PC) contribute to the maintenance of autoimmunity and inflammatory processes secreting pathogenic autoantibodies. These cells reside in specific survival niches in the bone marrow (BM) and inflamed tissues where they are resistant to immunosuppression and cytotoxic drugs. In this study the authors tested a new treatment approach aimed at depleting PC and, at the same time, preventing the generation of new autoreactive plasmablasts/PC that results from B cell-hyperreactivity. To this aimed the authors combined the PC-depleting drug bortezomib with an anti-CD20 antibody and analysed the PC-B cells dynamics in BM and spleen. Materials and methods Lupus-prone NZB/W mice were treated or not with a murine anti-CD20 antibody. After 1 week the mice were injected twice whit bortezomib to deplete short- and long-lived PC. To assess the grade of PC and B cell depletion (BCD) and to explore the dynamics of repopulation of the PC pool, the mice were killed at different time points after the treatment, and PC and B cells subsets were characterised by FACS and ELISPOT. Results Our results showed that after depleting short and long-lived PC with bortezomib these cells, including the autoreactive ones, were early regenerated. These results confirmed the hypothesis that new autoreactive PC can be generated from chronically activated B cells. Therefore, the authors treated NZB/W mice with bortezomib in combination with a murine anti-CD20 antibody. The group treated with the combination-therapy showed a heterogeneous grade of BCD more efficient in lymph nodes and spleen respect to BM. The mature, transitional and follicular B cells were efficiently depleted instead marginal zone, germinal center and prepro B cells were particularly resistant. Moreover, although in the mice treated with the combination therapy the repopulation of the PC pool was slower respect to bortezomib-treatment, the supply of newly generated autoreactive PC from hyperreactive-B cells was not completely suppressed. Conclusions Our data showed that PC are regenerated early after bortezomib mediated depletion. Moreover, anti-CD20 therapy was not able to completely prevent the differentiation of autoreactive B cells in long-lived autoreactive PC after depletion, maybe due to the incomplete BCD in NZB/W mice. These results shed new light on the dynamics and relationship between PC and B cells and emphasise a rationale for developing a combination therapy able to efficiently target both the compartment in autoimmune diseases.

Background Giant cell arteritis (GCA) and Takayasu arteritis (TAK) are an inflammation of the large arteries and their major branches. They have been considered distinct disorders based on their clinical features, age of onset and ethnic distribution. But the histopathology of arterial lesions and imaging studies suggest similar pathogenetic mechanisms in these diseases. Since the authors have recently shown disturbances in the B cell homeostasis in patients with active TAK the aim of this study was to analyse circulating B cell subpopulations in patients with GCA as compared to those with TAK. Methods Peripheral blood mononuclear cells from six patients with active TAK and six with inactive disease, 16 patients with GCA (eight with active and eight with inactive disease) and 12 healthy controls were analysed by flow cytometry for the expression of CD27, CD19, CD20, MHCII, CD3, CD8 and CD4. Plasma cell population is considered CD20−/CD19+/CD27+++. Newly generated plasmablasts express additionally MHC class II. CD19+/CD20+/CD27+ cells were considered as memory B cells and their CD27 low counterpart as naive B cells. Results Patients with active TAK and GCA showed significantly higher frequencies of plasma cells (among all CD19+ B cells) than in inactive patients (p=0.02 and 0.029, respectively) and healthy controls. The absolute plasma cell numbers were increased in TAK patients (p=0.0015) but not in GCA. Active TAK patients showed also elevated circulating plasmablasts in comparison with patients in remission (p=0.0053). The frequency of plasma cells in the blood correlated significantly with the disease activity according to the NIH criteria (r=0.73 and p=0.069). In contrast to TAK, active GCA patients exhibited a significant reduction of the memory B cell subset versus inactive patients (p=0.028). Disturbances of T cell subset were not observed. Conclusions The findings suggest different roles of B cells in the pathogenesis of TAK versus GCA. Circulating plasma cells/plasmablasts may serve as biomarker of disease activity in TAK and provide indication for a B cell and plasma cell directed therapy.

When activated, B cells can differentiate either into memory B cells, or into plasma cells, which secrete the B cells antibody at high rates. Plasma cells can be short-lived or long-lived. Long-lived plasma cells are maintained mostly in the bone marrow, in privileged survival niches, which are organised by dedicated stroma cells [1]. Long-lived plasma cells constitute a discrete type of immunological memory cells, providing humoral protection to recurrent pathogens. In immune-mediated diseases, like allergy or inflammatory rheumatic diseases, however, memory plasma cells can turn detrimental, constantly providing pathogenic antibodies. In systemic lupus erythematosus (SLE), autoreactive memory plasma cells, secreting antibodies against self antigens such as DNA, are readily detectable. These cells represent a therapeutic challenge. While short-lived plasma cells, respectable their precursors and their generation, are targeted by conventional immunosuppression with drugs such as cyclophosphamide, memory plasma cells do not respond, continue to secrete antibodies, and thus sustain persistence of disease [2,3]. New therapies, targeting memory plasma cells, have to be developed, to meet this challenge. Several different therapeutic strategies have been proposed so far.One of the more promising strategies aims at the inhibition of proteasomes, since this strategy has been shown to be effective to eliminate multiple myeloma cells, i.e. transformed plasma cells. In a first study, Neubert and collegues could show in 2008 that in the NZB/W mouse model of SLE, memory plasma cells are depleted from spleen and bone marrow by the proteasome inhibitor bortezomib (Velcade) [4]. Clinically, treated mice showed reduced nephritis and prolonged survival. Consequently, the groups of ReinhardVoll and our group initiated a clinical trial of bortezomib for the treatment of patients with SLE. Those patients had not responded to other treatments earlier and showed persistent autoantibody titers, indicating the presence of autoreactive memory plasma cells. Four patients with active SLE, previously treated with cyclophosphamide, received bortezomib (Velcade®) at doses of 1.3mg/m2 on days +1, 4, 8, 11 followed by a 10 days treatment-free interval for four cycles along with 20mg dexamethasone according to the approved myeloma protocol. Under proteasome inhibition, a significant clinical improvement was achieved in all patients associated with a reduction in lupus-specific autoantibodies. In addition, protective, vaccine-specific antibodies and total immunoglobulin levels dropped significantly, suggesting a depletion of bone marrow memory plasma cells secreting these antibodies.In all patients, like in the NZB/W mice, repeated treatment was needed for sustained efficacy, indicating that autoreactive memory plasma cells are continuously generated in chronic disease. Thus, a therapeutic regimen combining B cell depletion with memory plasma cells depletion might be preferable. First experiments in NZB/W mice showed a sustained effect of combination therapies depleting plasma cell precursors as well as fully differentiated plasma cells. In conclusion proteasome inhibition in SLE has demonstrated the therapeutic relevance of autoreactive memory plasma cells as such, and it constitutes a new therapeutic option, which has to be developed with respect to sustained effectivity, minimization of side effects and reduction of risk of infection.

Takayasu's arteritis (TA) is a large vessel vasculitis of unknown origin involving the aorta and its major branches. T cell mediated autoimmunity …

The current view holds that chronic autoimmune diseases are driven by the continuous activation of autoreactive B and T lymphocytes. However, despite the use of potent immunosuppressive drugs designed to interfere with this activation, the production of these autoantibodies often persists and contributes to progression of the immunopathology. In the present study, we analyzed the lifespan of (auto)antibody-secreting cells (ASC) in the spleens of NZB/W mice, a murine model of human systemic lupus erythematosus (SLE). The number of splenic ASC increased in mice aged 1–5 months and became stable thereafter. Less than 60% of the splenic antibody-secreting cells were short-lived plasmablasts, whereas 40% were non-dividing, long-lived plasma cells with a half-life of more than 6 months. In NZB/W mice and D42 immunoglobulin heavy chain 'knock-in' mice, we found that a fraction of DNA-specific plasma cells were also long-lived. Although antiproliferative immunosuppressive therapy eliminates short-lived plasmablasts, long-lived plasma cells can survive and continue to produce (auto)antibodies. Thus, long-lived, autoreactive plasma cells are a relevant target for researchers aiming to develop curative therapies for autoimmune diseases.

According to the current view, chronic AID are driven by the continuous activation of autoreactive B and T lymphocytes. However, despite the use of potent immunosuppressive drugs, these patients exhibit continued autoantibody production, which often contributes to progression of the AID. To elucidate this problem, we analyzed the lifespan of cells secreting (auto)antibodies (Ab) in NZB/W mice. The number of Ab-secreting cells in splenic tissue increased from ages 1 to 5 months and became stable thereafter. Later, some 60% of the total Ab-secreting cell compartment consisted of dividing, short-lived plasmablasts (SLPB), whereas 40% consisted of nondividing, long-lived plasma cells (LLPC) with a half-life of more than 6 months. Although high-dose cyclophosphamide eliminated SLPB, LLPC survived and continued to produce (auto)Ab. This explains why treatment with cyclophosphamide (CY) does not result in the complete disappearance of antinuclear Ab. Pathogenic anti-dsDNA Ab usually vanish after this treatment, indicating that they are generated by proliferating SLPB. However, anti-dsDNA Ab can be resistant to CY in refractory SLE. We treated six refractory SLE patients by autologous stem cell transplantation (ASCT) using CY and ATG as the mobilization regimen. ASCT was performed using purified CD34+ stem cells. The patients have now been followed for 5–70 months. Initially, persistent anti-dsDNA disappeared within 3 months. Likewise, the ANA became negative except in one case. That patient continued to exhibit anti-Ro/SSA and anti-La/SSB, the titers of which decreased continuously and were undetectable after 17 months. This emphasizes the enormous lifespan of residual plasma cells after interrupting the supply of new cells from the dividing SLPB compartment. In conclusion, the aforementioned ASCT protocol can lead to depletion of autoreactive LLPC. Since ASCT may not always be able to eliminate autoreactive LLPC, these cells are potential targets for autoimmune disease therapy.

Chronic inflammatory diseases (CID) are emerging disorders which do not only affect specific organs with respective clinical symptoms but can also affect various aspects of life, such as emotional distress, anxiety, fatigue and quality of life. These facets of chronic disease are often not recognized in the therapy of CID patients. Furthermore, the symptoms and patient-reported outcomes often do not correlate well with the actual inflammatory burden. The discrepancy between patient-reported symptoms and objectively assessed disease activity can indeed be instructive for the treating physician to draw an integrative picture of an individual’s disease course. This poses a challenge for the design of novel, more comprehensive disease assessments. In this mini-review, we report on the currently available patient-reported outcomes, the unmet needs in the field of chronic inflammatory diseases and the challenges of addressing these.

Summary The persistence of long-lived memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, which are provided in niches organized by stromal cells. Using an ex vivo system in which we supply the known survival signals, direct cell contact to stromal cells, and the soluble cytokine a proliferation-inducing ligand (APRIL), we have elucidated the critical signaling pathways required for the survival of long-lived plasma cells. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to critical inactivation of Forkhead-Box-Protein O1/3 (FoxO1/3) and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Accordingly, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, by the nuclear factor κB (NF-κB) pathway, blocks activation of the endoplasmic-reticulum-stress-associated initiator caspase 12. Thus, stromal-cell-contact-induced PI3K and APRIL-induced NF-κB signaling provide the necessary and complementary signals to maintain bone marrow memory plasma cells., Graphical Abstract, Highlights • Description of an in vitro niche for the cultivation of primary murine plasma cells • Stromal cell contact and APRIL are essential for survival of bone marrow plasma cells • Stromal-cell-contact-induced PI3K signaling inhibits FoxO1/3 and caspase 3 activation • APRIL-induced NF-κB signaling prevents caspase 12 activation, In this study, Cornelis et al. address the molecular mechanisms underlying the survival of murine memory plasma cells in the bone marrow. The authors provide evidence that direct contact to stromal cells and exogenous APRIL provide resilience to mitochondrial and endoplasmic stress, respectively, synergistically promoting plasma cell survival.