Laleh Khodadadi

Autoantibodies from long-lived 'memory' plasma cells of NZB/W mice drive immune complex nephritis.

Objectives We have previously shown that both short- and long-lived plasma cells (PCs) significantly contribute to autoantibody production in NZB/W mice as a model of lupus nephritis. The aim of this study was to determine the role of autoreactive long-lived (memory) PCs refractory to immunosuppression and B cell depletion in the pathogenesis of systemic lupus erythematosus. Methods Splenic CD138+ antibody-secreting cells (ASCs) from >6-month-old NZB/W mice with high titres of anti-dsDNA autoantibodies or from Balb/c mice 5 days after secondary immunisation with ovalbumin (OVA) were adoptively transferred to immunodeficient Rag1−/− mice, in which the development of nephritis was investigated by measuring proteinuria. Total IgG and IgM as well as anti-dsDNA and anti-OVA antibody levels were followed up by ELISA. After 21 weeks the recipient mice were sacrificed so that PCs in spleen and bone marrow could be analysed using ELISPOT and flow cytometry and renal immunohistology performed. Results The adoptive transfer of NZB/W and anti-OVA ASCs resulted in the continuous generation of anti-dsDNA antibodies and anti-OVA antibodies, respectively, exclusively by long-lived PCs that had homed to the spleen and bone marrow of recipient Rag1−/− mice. Rag1−/− mice generating autoantibodies including anti-dsDNA had reduced survival, proteinuria and immune complex nephritis with C1q, C3, IgG and IgM deposits 21 weeks after transfer. Conclusions These findings demonstrate that autoantibodies exclusively secreted by long-lived (memory) PCs contribute to autoimmune pathology and should be considered as candidate targets for future therapeutic strategies.

Bortezomib Plus Continuous B Cell Depletion Results in Sustained Plasma Cell Depletion and Amelioration of Lupus Nephritis in NZB/W F1 Mice.

Long-lived plasma cells (LLPCs) are an unmet therapeutic challenge, and developing strategies for their targeting is an emerging goal of autoantibody-mediated diseases such as systemic lupus erythematosus (SLE). It was previously shown that plasma cells can be depleted by agents such as bortezomib (Bz) or by blocking LFA-1 and VLA-4 integrins. However, they regenerate quickly after depletion due to B cell hyperactivity in autoimmune conditions. Therefore, we compared different therapies for the elimination of LLPCs combined with selective B-cell targeting in order to identify the most effective treatment to eliminate LLPCs and prevent their regeneration in lupus-prone NZB/W F1 mice. Methods NZB/W F1 mice were treated with: 1) anti-CD20, 2) anti-CD20 plus bortezomib, 3) anti-CD20 plus anti-LFA-1/anti-VLA-4 blocking antibodies, 4) anti-CD20 plus bortezomib and anti-LFA-1/anti-VLA4 blocking antibodies. Short- and long-lived plasma cells including autoreactive cells in the bone marrow and spleen were enumerated by flow cytometry and ELISPOT seven days after treatment. Based on these data in another experiment, mice received one cycle of anti-CD20 plus bortezomib followed by four cycles of anti-CD20 therapy every 10 days and were monitored for its effect on plasma cells and disease. Results Short-lived plasma cells in bone marrow and spleen were efficiently depleted by all regimens targeting plasma cells. Conversely, LLPCs and anti-dsDNA-secreting plasma cells in bone marrow and spleen showed resistance to depletion and were strongly reduced by bortezomib plus anti-CD20. The effective depletion of plasma cells by bortezomib complemented by the continuous depletion of their precursor B cells using anti-CD20 promoted the persistent reduction of IgG anti-dsDNA antibodies, delayed nephritis and prolonged survival in NZB/W F1 mice. Conclusions These findings suggest that the effective depletion of LLPCs using bortezomib in combination with a therapy that continuously targeting B cells as their precursors may prevent the regeneration of autoreactive LLPCs and, thus, might represent a promising treatment strategy for SLE and other (auto)antibody-mediated diseases.

Analyzing pathogenic (double-stranded (ds) DNA-specific) plasma cells via immunofluorescence microscopy

IntroductionWhile protective plasma cells (PCs) are an important part of the individual’s immune defense, autoreactive plasma cells such as dsDNA-specific plasma cells contribute to the pathogenesis of autoimmune diseases like systemic lupus erythematosus (SLE). However, the research on dsDNA-specific plasma cells was restricted to the ELISpot technique, with its limitations, as no other attempt for identification of dsDNA-reactive plasma cells had been successful.MethodsWith improved fluorochrome labeling of dsDNA, removal of DNA aggregates, and enhanced blocking of unspecific binding, we were able to specifically detect dsDNA-reactive plasma cells by immunofluorescence microscopy.ResultsVia this novel technique we were able to distinguish short-lived (SLPCs) and long-lived (LLPCs) autoreactive plasma cells, discriminate dsDNA-specific plasma cells according to their immunoglobulin class (IgG, IgM, and IgA) and investigate autoreactive (dsDNA) and vaccine-induced ovalbumin (Ova) plasma cells in parallel.ConclusionsThe detection of autoreactive dsDNA-specific plasma cells via immunofluorescence microscopy allows specific studies on pathogenic and protective plasma cell subsets and their niches, detailed evaluation of therapeutic treatments and therefore offers new possibilities for basic and clinical research.

Proteasome inhibition with bortezomib induces a therapeutically relevant depletion of plasma cells in SLE but does not target their precursors

Long‐lived plasma cells (PCs) not only provide protective humoral immunity, they are also an essential component of the autoreactive immunologic memory that may drive chronic immune responses in systemic autoimmunity, such as systemic lupus erythematosus (SLE). The therapeutic relevance of their targeting has been demonstrated in preclinical models and severe, treatment‐refractory cases of autoimmune diseases using the proteasome inhibitor bortezomib. Herein, we describe in detail the dynamic serologic changes and effects on immune effector cells in eight SLE patients receiving a median two cycles of 1.3 mg/m2 intravenous bortezomib. Upon proteasome inhibition, immunoglobulin levels gradually declined by ∼30%, associated with a significant reduction of autoantibodies, and serum complement whereas B‐cell activation factor levels increased. While proteasome inhibition was associated with a significant depletion of short‐ and long‐lived PCs in peripheral blood and bone marrow by ∼50%, including those with a distinctly mature CD19− phenotype, their precursor B cells and T cells largely remained unaffected, resulting in a rapid repopulation of short‐lived PCs after bortezomib withdrawal, accompanied by increasing autoantibody levels. Collectively, these findings identify proteasome inhibitors as a promising treatment option for refractory SLE, but also indicate that PC depletion needs to be combined with targeted B‐cell therapies for sustained responses in systemic autoimmunity.

S1D:6 Targeting plasma cells and their precursors by immunoablation versus bortezomib plus rituximab in systemic lupus erythemtosus

Purpose To investigate the therapeutic relevance of targeting long-lived plasma cells (PC), which contribute to the chronicity of SLE through continuous secretion of pathogenic antibodies, using immunoablation with antithymocyte-globulin (ATG) in the context of haematopoietic stem cell transplantation (HSCT) or proteasome inhibition with Bortezomib. Methods Prospective analysis of outcome in 10 SLE patients after receiving autologous HSCT between 1998 and 2012 and 8 SLE patients after receiving a median 2 cycles (range 1–4) of Bortezomib 1.3 mg/m2 between 2009 and 2012 at the Charité – University Medicine Berlin. Multiparametric flow cytometry was applied to characterise peripheral blood or bone marrow PC subsets and B cells. Autoantibodies and vaccine titres were investigated with ELISA. Results In all HSCT treated patients clinical remissions (SLEDAI <3) were achieved, accompanied by a complete normalisation of anti-dsDNA antibody titres (92.6% reduction) and a significant reduction of antinuclear antibodies and vaccine titres (measles 82.3%). Peripheral blood B and PCs were virtually absent and bone marrow PCs largely depleted (97.6% reduction, n=1) shortly after HSCT and regenerating B cells almost exclusively displayed a naïve phenotype. Upon proteasome inhibition, clinical improvements were associated with a significant reduction of anti-dsDNA autoantibodies (69.3%) and vaccine titers (measles 32.5%). While B cell number/phenotype remained stable, both bone marrow and circulating PC were significantly reduced (~50%) but rapidly regenerated after proteasome inhibition, which could be prevented by additional rituximab therapy in one patient applied. Conclusions Although less prominent compared to HSCT, proteasome inhibition with Bortezomib promoted a therapeutically relevant PC depletion in refractory SLE. Nevertheless, for sustained responses, PC depletion needs to be combined with therapeutic strategies targeting their precursor B cells, e.g. with rituximab, as indicated by our preclinical studies in murine lupus.

A6.35 Differential B and plasma cell homing mechanisms in inflamed kidneys of NZB/W F1 mice

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+ naïve 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 naïve B cells whereas plasmablast and plasma cell infiltration seems to be mediated by different mechanisms yet unclear mechanism.

A8.35 Long-lived plasma cell dynamics in autoimmunity: from the homeostasis of the immunological memory to new therapeutic challenges

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.

THU0041 Autoreactive Long-Lived Plasma Cells in Nzb/W Mice and their Regeneration

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

SAT0168 Targeting of long-lived plasma cells in autoimmune NZB/W mice

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