Adriano Taddeo

The proteasome inhibitior bortezomib depletes plasma cells and ameliorates clinical manifestations of refractory systemic lupus erythematosus

Objectives To 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). Methods Twelve 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. Results Upon 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. Conclusions These 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.

The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity

The protein tyrosine phosphatase PTP1B regulates co-receptor signaling on B cells and thus controls B cell autoimmunity.

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.

Selection and depletion of plasma cells based on the specificity of the secreted antibody

Plasma cells (PCs) are antibody-secreting cells (ASCs) that play an important role in immunity and autoimmunity. There is an urgent need to develop efficient and simple methods for analyzing the biology and function of PCs at the single-cell level. Here, we describe a method for the selective labeling of viable cells secreting antibodies of a given specificity. We demonstrate that these labeled PCs can be isolated alive or induced to commit suicide in the presence of complement. The method is based on affinity matrix (AM) technology that we developed previously [1, 2] and on a method to enrich mutants of a hybridoma cell line [3]. Antigen specificity is provided by the conjugation of an antibody or a F(ab) fragment recognizing the surface markers CD138 or CD44 on PCs with the antigen of interest. The matrix is used to coat the surface of PCs with the antigen of interest — in this case, ovalbumin (OVA) or acetylcholine receptor (AChR). Secreted antibodies of the respective specificity bind to the antigen attached via the cell-surface AM of the secreting cells (Fig. 1A). The secreted antibody bound to the ASCs can then be identified and stained by immunofluorescence, using an anti-Ig isotype antibody. Alternatively, in the presence of complement, they can be induced to commit suicide, lysed by their own antibodies. The anti-CD138-OVA or anti-CD44OVA AM strongly labels the surface of hybridoma cells, inducing an approximately 104 shift in mean fluorescence intensity when stained with antigenspecific antibody (Supporting Information Fig. 1). When the anti-OVA hybridoma cells are coated with the anti-CD138OVA AM and incubated at 37°C for 10 min, the antigen in the matrix is efficiently labeled with secreted antibodies with a 58-fold increase in mean fluorescence intensity when the cells are stained extracellularly with fluorescent isotypespecific antibody (Supporting Information Fig. 2). The sensitivity of the method was analyzed by spiking populations of CFSE-labeled 4C9 hybridoma cells secreting OVA-specific antibodies into a population of X63-Ag6.653 myeloma cells at ratios of 1:1 to 1:10 000. The OVA-specific hybridoma cells were selectively labeled by secreted anti-OVA-IgG at all ratios analyzed (Fig. 1B). Decreasing the permeability of the incubation medium (see Supporting Information for details) limited the diffusion of the antibody of interest (cross-feeding); this was used to avoid unspecific labeling in culture, with a specific:unspecific cell ratio of 1:1 (Fig. 1C) and 2:1 (not shown). The AM also allowed the distinction of specific PCs with different IgG subclasses when hybridoma cells producing IgG1 or IgG2b anti-OVA antibodies were mixed (Supporting Information Fig. 3). Moreover, the AM can be used to rapidly select and sort viable anti-OVA-producing hybridoma cells in a cell line with partially lost OVA antibody production due to long-term culture, obviating the need for laborand time-intensive subcloning and screening for reestablishment of the monoclonal culture (Supporting Information Fig. 4). This AM technology was also used ex vivo for the identification and sorting of viable antigen-specific PCs from the lymphoid organs of BALB/c mice immunized with OVA. Spleen and bone marrow PCs marked with the matrix (anti-CD138OVA) were incubated for 30 min at 37°C to allow for antibody secretion. The secreted anti-OVA antibody captured by the matrix was labeled using an anti-mouse IgG1 antibody. As shown in Figure 1D, only OVAspecific PCs were labeled by the secreted antibody, as determined by the acquisition of IgG1 expression on their surfaces and by their intracellular OVA binding. Sorting the PCs based on the secreted IgG1 yielded an OVA-specific PC population with a purity >75% (Supporting Information Fig. 5). The AM technology was also used to selectively deplete PCs secreting antibodies of a given specificity with complement present in the culture medium (Fig. 2). As proof of concept, OVAsecreting hybridoma cells were incubated with the AM and cultured for 30 min at 37°C in medium supplemented with 10% guinea pig complement. After exclusion of dead cells by DAPI staining, viable cells were counted by flow cytometry. AntiOVA-specific hybridoma cells were specifically lysed when the cells were coated with the matrix, while the CD138-positive X63-Ag6.653 myeloma cells were not significantly depleted (Fig. 2A) unless antiOVA antibodies were added to the culture (Supporting Information Fig. 6). The specificity of the AM technology was confirmed by using a mixture of specific anti-OVA-secreting hybridoma cells and ASCs of unrelated specificity. As shown in

A3.26 Proteasome inhibition with bortezomib in refractory SLE inhibits type I interferon and depletes plasma cells but does not inhibit their regeneration

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/m2, 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.

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.

06.14 Proteasome inhibition with bortezomib in sle promotes therapeutically relevant depletion of short- and long-lived plasma cells but does not prevent their regeneration

Objectives We previously identified long-lived plasma cells (PCs) are an essential component of the pathogenic immunologic memory, which is resistant to immunosuppressive and B cell depleting therapies and contributes to the chronicity of SLE. We recently demonstrated that their targeting with the proteasome inhibitor bortezomib resulted in therapeutically relevant plasma cell depletion in refractory cases of SLE. Here we investigated in detail the cellular and serologic responses of bortezomib treatment. Methods Eight patients received a median of two 21-day cycles of intravenous bortezomib 1.3mg/m2 as induction therapy for active SLE. Disease activity was assessed using the SLEDAI-2K score. Serum concentrations of anti-double-stranded DNA (anti-dsDNA), vaccine-induced antibodies and BAFF levels were monitored. Flow cytometry was performed to analyse peripheral blood B cells, T cells and PCs and Siglec-1 expression on monocytes as surrogate marker for type-I interferon (IFN). Results Upon proteasome inhibition, serum levels for anti-double-stranded (ds)DNA (~60%) and vaccine-induced antibodies for Measels, Mumps, TT (~30%) significantly declined, complement levels and serum BAFF levels significantly increased and SLEDAI significantly decreased (p=0.014). The level of bone marrow PCs decreased by 50%, affecting both CD19+ and CD19- CD138/CD38 expressing PCs. While number and phenotype of peripheral blood T- and B cells remained unaffected, proteasome inhibition was associated with a significant depletion of HLA-DR+ (p=0.024), HLA-DR- (p=0.038) and IgA+ (p=0.032) PCs, but their regeneration was fast. Within ten days from the last bortezomib injection, their numbers significantly increased (beta=2.1; 95% CI: 0.5, 3.7; p=0.012), an effect that was more pronounced in HLA-DR+ short-lived than in HLA-DR- long-lived PCs. Overall, the magnitude of increase over time did not significantly decrease. Siglec-1 expression on monocytes significantly declined. Conclusions These findings identify proteasome inhibitors as a promising novel treatment option for patients with refractory SLE by targeting short- and long-lived PCs and type I IFN activity. Bortezomib efficiently induces short-term remissions but would require additional B cell directed treatment approaches to inhibit PC regeneration from their precursors for sustained efficacy.

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.

A8.28 Depletion of autoantibody-secreting plasma cells based on the specificity of the secreted antibody

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.

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.