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ObjectivesTo investigate whether bortezomib, a proteasome inhibitor approved for treatment of multiple myeloma, induces clinically relevant plasma cell (PC) depletion in patients with active, refractory systemic lupus erythematosus (SLE).MethodsTwelve patients received a median of two (range 1–4) 21-day cycles of intravenous bortezomib (1.3 mg/m2) with the coadministration of dexamethasone (20 mg) for active SLE. Disease activity was assessed using the SLEDAI-2K score. Serum concentrations of anti–double-stranded DNA (anti-dsDNA) and vaccine-induced protective antibodies were monitored. Flow cytometry was performed to analyse peripheral blood B-cells, PCs and Siglec-1 expression on monocytes as surrogate marker for type-I interferon (IFN) activity.ResultsUpon proteasome inhibition, disease activity significantly declined and remained stable for 6 months on maintenance therapies. Nineteen treatment-emergent adverse events occurred and, although mostly mild to moderate, resulted in treatment discontinuation in seven patients. Serum antibody levels significantly declined, with greater reductions in anti-dsDNA (∼60%) than vaccine-induced protective antibody titres (∼30%). Bortezomib significantly reduced the numbers of peripheral blood and bone marrow PCs (∼50%), but their numbers increased between cycles. Siglec-1 expression on monocytes significantly declined.ConclusionsThese findings identify proteasome inhibitors as a putative therapeutic option for patients with refractory SLE by targeting PCs and type-I IFN activity, but our results must be confirmed in controlled trials.

Background and objectives Long-lived plasma cells (LLPCs) producing pathogenic antibodies are an important factor in autoimmune diseases maintenance and relapse. The therapeutic options available so far for targeting these cells (e.g. autologous stem cell transplantation, bortezomib) have the major disadvantage of eliminating the protective humoral immunity with the obvious caveats regarding a higher risk of infection. Therefore the development of new therapeutic tools for the selective depletion of LLPCs secreting pathogenic antibodies would be a great step forward. Material and methods We synthetized an antigen affinity matrix (AM) conjugating a plasma cell specific antibody (i.e. anti-CD138) with the antigen/autoantigen-of-interest (i.e. Ovalbumin or Acetylcholine-Receptor, AChR). We performed proof-of-concept experiments using the anti-CD138-ovalbumin using: 1) anti-ovalbumin secreting hybridoma cells and 2) spleen cells from ovalbumin-immunised mice. Moreover, we used the anti-CD138-AChR AM for the ex-vivo depletion of splenic pathogenic plasma cells isolated from mice with experimental autoimmune myasthenia gravis (EAMG). Results Antibodies secreted by ovalbumin-specific plasma cells could bind the ovalbumin delivered at surface of the plasma cell by the AM. In this way, anti-ovalbumin-specific hybridoma cells and splenic plasma cells were induced to commit suicide in presence of complement. Conversely, unrelated plasma cells (i.e. secreting antibodies against other antigens) were not lysed confirming the specificity of the method. Importantly, pathogenic plasma cells secreting anti-AChR-autoantibodies from the spleen of mice with EAMG can be eliminated using an anti-CD138-AChR-construct, suggesting that the AM technology can be used for the specific depletion of autoantibodies secreting plasma cells. Conclusion Here we show that the AM technology could represent the first available tool for the depletion of (auto)antigen-specific plasma cells without affecting the protective plasma cells. We are currently testing the therapeutic efficacy of this AM in murine models of autoimmune disease.

NZB/W mice spontaneously develop a lupus-like disease leading to lethal immune complex-mediated nephritis. Disease manifestation is accompanied by inflammatory infiltration of the kidneys by lymphocytes. Here, we show that the immigration of B cells is mediated via CXCR5 whereas plasma cell infiltration is differently. Histology was used to analyse the distribution of lymphocyte subsets and chemokines within inflamed NZB/W kidneys, and flow cytometry was used for the qunatification of, the phenotyping and chemokine receptor expression of particular lymphocyte subsets. Our data show that kidney-infiltrating B cells accumulate within small, follicle-like structures of the kidney, whereas plasma cells and plasmablasts are scattered in conglomerates of several cells throughout the whole organ. B cells expressing the chemokine receptor CXCR5 can be found in areas of high CXCL13 concentration. In contrast plasma cells and plasmablasts express low levels of CXCR5 but high levels of CXCR3 and CXCR4, the ligands for CXCL10 and CXCL12 respecitvally known to be overexpressed in inflammatory tissue and bone marrow which might explain the different distribution pattern. Interestingly, the kidney-infiltrating B cell population contains 50% IgD/IgM+ naive cells and also includes smaller proportions of cells exhibiting a phenotype of CD93 + /CD23 + /- T1/T2/3 immature B cells. These data suggest that B cells accumulate in the kidneys through homing mechanisms involving CXCR5/CXCL13 attracting primarily naive B cells whereas plasmablast and plasma cell infiltration seems to be mediated by different mechanisms yet unclear mechanism.

Background and objectives Plasma cell depletion with bortezomib can prevent the development of lupus nephritis and prolong survival in NZB/W mice. Here, we studied the effect of the immunisation with OVA in combination with bortezomib on the disease in NZB/W mice. Methods NZB/W F1 female mice 16 week-old (early disease stage) were divided into 3 groups as follows: 1. Non-immunised mice as a control group, 2. Immunised mice; mice immunised with alum-precipitated OVA i.p and boosted 3 weeks after primary immunisation, 3. Immunised mice as group 2 receiving two shots of bortezomib (0.75 mg/kg, i.v) at 48 and 12 h before secondary immunisation. The numbers of anti-dsDNA antibody secreting cells (ASCs) were assessed in spleen and bone marrow by ELISPOT one month after secondary immunisation. Moreover we investigated serologic parameters including anti-OVA and anti-dsDNA antibody levels by ELISA as well as proteinuria and survival rate. Results Immunised mice (group 2) developed proteinuria, which occurred earlier and stronger than in the non-immunised mice and in mice additionally treated with Bz before booster immunisation (group 3). Survival rate of immunised mice was reduced in comparison to group 3 treated with Bz while group 1 showed the longest survival. Autoantibody levels in group 2 and 3 were higher than in the control group 1. Mice immunised with OVA showed a tendency to higher numbers of IgM anti-dsDNA ASCs in the bone marrow and spleen than in non-immunised mice while IgG anti-dsDNA ASCs counts analysed in mice immunised with OVA and treated with Bz, were comparable with their counts in non-immunised mice. The Bz treatment before the secondary OVA immunisation resulted in lower anti-OVA antibody levels than in immunised mice without Bz treatment. Conclusion Immunisation with alum-precipitated OVA accelerates autoimmunity in NZB/W mice. This effect can be ameliorated by plasma cell depletion with bortezomib.

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 B cells are playing a major role in granulomatosis with polyangiitis (GPA, formerly known as Wegener’s disease). This is reflected by autoantibodies directed against neutrophil granular encymes (ANCA) as well as in the success of B cell depleting therapies. Whether mucosal plasma cells play a role in ENT-involvement is yet unknown. IgA-ANCA are found in about 30% of GPA patients and more often in patients with ENT-GPA (Kelley et al). For a better understanding of the possible role of plasma cells in GPA we analysed B cells subsets in the peripheral blood of patients and found major changes correlating with disease activity (BVAS). Methods 18 patients with GPA (11 with active disease, 7 in remission) were analysed by flow cytometry and compared to 17 healthy donors and 6 patients with systemic sclerosis. Stainings for CD19, 20, 27, IgD, IgA and MHCII were performed and analysed by FlowJo-software. The study was approved by the Charite ethics committee and all patients signed informed consent. Statistical analysis was performed using GraphPadPrism. Results : Marked differences (p = 0.0018) were found regarding the number and frequency of plasmablasts/plasma cells in patients with active disease (6.4 ± 5.06/µl) as compared to patients in remission (2.5 ± 1.6/µl) or healthy donors (2.3 ± 1.2/µl). In patients with GPA a significant higher number of the plasma cells produced IgA as compared to healthy controls (p = 0.0028). The same was true in patients with systemic sclerosis. So far, no difference regarding the IgA plasma cells in the blood was observed between patients with ENT-GPA and patients with systemic GPA (sGPA), while sGPA patients tend to have more plasma cells in the blood. The number of plasma cells and IgA plasma cells correlate with disease activity (r = 0.9135, p Conclusion The number of plasma cells in active GPA is increased. This implies a central role of plasma cells in the pathogenesis of GPA. A high frequency of these plasma cells is producing IgA, which could play a role in ENT-involvement. So far now diffenerce could be observed between ENT-GPA and sGPA but further studies and more detailed studies including the analysis of ENT biopsies are needed to further understand their role in disease pathogenesis.

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 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.