Plasma Cell Survival Research Articles

TACI-dependent APRIL signaling maintains autoreactive B cells in a mouse model of systemic lupus erythematosus.

Autoantibodies contribute to the development of systemic lupus erythematosus (SLE). APRIL (a proliferation-inducing ligand), a member of the TNF superfamily, regulates plasma-cell survival and binds to TACI (transmembrane activator CAML interactor) and BCMA (B-cell maturation antigen). We previously showed that APRIL blockade delayed disease onset in lupus-prone mice. In order to evaluate the role of APRIL receptors in the development of SLE, APRIL, TACI, BCMA, or double TACI.BCMA null mutations were introduced into the Nba2.Yaa (Y-linked autoimmune acceleration) spontaneous lupus mouse model. Mortality as a consequence of glomerulonephritis (GN) was reduced in Nba2.APRIL-/- .Yaa, Nba2.TACI-/- .Yaa and double-KO mice compared with Nba2.Yaa mice and correlated with lower levels of circulating antibodies, while splenic populations remained unchanged. In contrast, the appearance of symptoms was accelerated in BCMA-deficient mice, in which TACI signaling was increased. Finally, lupus-prone mice deficient for the APRIL-TACI axis produced less pathogenic antibodies and developed less GN. Disease reduction was attributed to impaired T-independent type 2 responses when the APRIL-TACI signaling axis was disrupted. Collectively, our results have identified and confirmed APRIL as a new target involved in B-cell activation, in the maintenance of plasma cell survival and subsequent increased autoantibody production that sustains lupus development in mice.

Impact of CXCR4/CXCL12 Blockade on Normal Plasma Cells In Vivo.

Plasma cells (PCs) are a major source of alloantibody in transplant patients and are resistant to current therapy. Because receptor-ligand interactions in stromal microenvironments play important roles in the localization, development, and survival of normal PCs, we hypothesized that interfering with CXCR4/CXCL12 interactions with plerixafor might cause PC depletion and enhance the efficacy of the proteasome inhibitor bortezomib. PCs in mouse spleen, bone marrow, and peripheral blood demonstrated CXCR4 expression. We then treated with plerixafor in doses ranging from 240 μg/kg in a single dose to a 1-mg/kg daily dose for 10 days. CXCR4/CXCL12 blockade with plerixafor resulted in increased mobilization of PCs into the peripheral blood. Splenectomy completely abrogated this effect, suggesting that all plerixafor-mobilized cells were from the spleen. The total number of PCs in the spleen and marrow remained constant despite treatment with plerixafor. Bortezomib caused a reduction in PCs, but adding plerixafor did not increase killing. We conclude that CXCR4/CXCL12 interactions are important for the retention of a subpopulation of PCs in the spleen, but this interaction has minimal effect on PCs in the marrow. The lack of enhancement of bortezomib-mediated depletion suggests that factors other than CXCR4/CXCL12 interactions are responsible for drug resistance.

The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis.

Plasma cells (PCs) are exposed to intense endoplasmic reticulum (ER) stress imposed by enormous rates of immunoglobulin (Ig) synthesis and secretion. Therefore, protein homeostasis is crucial for the survival of PCs, but its molecular mechanism remains largely unknown. Here, we found marked overexpression of FK506-binding protein 13 (FKBP13) in long-lived PCs from autoimmune mice and investigated its function using a plasmacytoma cell line secreting IgA. FKBP13 expression was induced largely in the lumen of ER in response to treatment with an ER stressor tunicamycin or overexpression of an adaptive unfolded protein response (UPR) protein X-box binding protein 1 (XBP1). Silencing of FKBP13 expression led to induction of molecules involved in the terminal UPR and ER stress-associated apoptosis. FKBP13 interacted with Ig, facilitated its ubiquitination, and lowered the extent of ER stress. FKBP13 overexpression caused a significant reduction in secreted IgA in plasmacytoma cells, and FKBP13 knockdown exerted an opposite effect. Rapamycin interfered with the interaction between FKBP13 and IgA and enhanced the amount of secreted IgA. Importantly, the level of FKBP13 was inversely correlated with the amount of secreted antibody in long-lived PCs from autoimmune mice. These results suggest that FKBP13 is a marker of long-lived PCs and a component of XBP1-dependent ER protein homeostasis. FKBP13 is likely to act as a molecular chaperone that delivers misfolded ER clients, including Ig, to ER-associated degradation, so reducing proteotoxic stress on the PC. Our data reveal a novel cytoprotective role for FKBP13 in long-lived PCs occurring at the expense of antibody production.

Decreased Expression of B cell Maturation Antigen in Patients with Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous group of diseases characterized by hypogammaglobulinemia, leading to recurrent infections. Defect in maturation of B cells into functional plasma cells is the most principal defect in CVID. B cell maturation antigen (BCMA) is an essential molecule for survival of long-lived plasma cells. The aim of this study is to evaluate BCMA expression on B cells as well as count peripheral blood plasmablast cells in CVID patients compared with healthy individuals. Blood samples were collected from 15 CVID patients and 15 age- and sex-matched normal controls. Genomic RNA was extracted from peripheral blood mononuclear cell samples using an RNX-Plus Kit. cDNA was synthesized from extracted RNA using a RevertAid First Strand cDNA Synthesis Kit. Synthesized cDNA was used as a template for reverse transcription polymerase chain reaction (RT-PCR) to measure BCMA gene expression by Maxima SYBR Green/ROX qPCR Master Mix Kit (Thermo Scientific). Flow cytometry was use...

Targeting Ongoing DNA Damage in Multiple Myeloma. Effects of Different Inhibitors of the DNA Damage Response on Plasma Cell Survival

Targeting Ongoing DNA Damage in Multiple Myeloma. Effects of Different Inhibitors of the DNA Damage Response on Plasma Cell Survival

P761 Gene expression differences between Crohn's disease aphthous ulcers and healthy Peyer's patches highlights potential treatment strategies

Background: The earliest macroscopic lesion in Crohn's disease (CD) is the aphthous ulcer, which overlies Peyer's patches and lymphoid follicles. Our aim was to characterize differences in gene expression of aphthous ulcers and Peyer's patches. Methods: Biopsies were obtained from the terminal ileum of 12 patients (6 with CD, 6 healthy controls). Aphthous ulcers and adjacent unaffected mucosa were obtained from CD patients, and Peyer's patches and adjacent mucosa from the controls. All patients were medication-free, except one. RNA was extracted using Qiagen kits. NextSeq 500 libraries were constructed using NextSeq 500/550 High output kits (Illumina) in a 150 bp paired-end format. Transcripts were assessed for quality using FASTQC, trimmed using Trimmomatic, and aligned to the human reference genome using subread mapper. Fragment counts were obtained using featureCount, and expression values normalized using the trimmed mean of M-values normalization method (TMM). Differential gene expression analyses were performed using generalized linear models in edgeR. Cell-specific gene expression was determined using ImSig. Results: We obtained 36 million tags per sample, 87% were retained for downstream processing, and 93% of these mapped to the human genome. 685 genes were significantly differentially expressed between aphthous ulcers and Peyer's patches, all were upregulated in aphthous ulcers. Differential gene expression analysis revealed 34 pathways that were upregulated in aphthous ulcers relative to Peyer's patches. Receptors for the constant region of immunoglobulin (Ig) G were represented in 13 of these pathways. Expression of the high affinity FCGR1A (CD64), and low affinity FCGR3A (CD16), FCGR2A and FCGR2C (CD32), was significantly upregulated in aphthous ulcers (p=1.14E-12), but not the inhibitory FCGR2B (CD32B). Other pathways that were highly upregulated in aphthous ulcers included those involved in responding to bacteria, leukocyte chemotaxis, inflammatory response, and creation of C2 and C4 activators. ImSig, which is capable of using transcriptome data to indicate cell types and their activation state, revealed that core marker genes for plasma cells were overrepresented in aphthous ulcers relative to Peyer's patches and unaffected/normal mucosa. Conclusions: The most significantly upregulated Fc gamma receptors in aphthous ulcers are the major receptors expressed on monocytes/macrophages, which are involved in regulating innate and adaptive immune responses, as well as plasma cell survival (FCGR2B), making them an attractive target for immunotherapy.

Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

Pre-existing antibodies (Abs) to AAV pose a critical challenge for the translation of gene therapies. No effective approach is available to overcome pre-existing Abs. Given the complexity of Ab production, overcoming pre-existing Abs will require broad immune targeting. We generated a mouse model of pre-existing AAV9 Abs to test multiple immunosuppressants, including bortezomib, rapamycin, and prednisolone, individually or in combination. We identified an effective approach combining rapamycin and prednisolone, reducing serum AAV9 Abs by 70%–80% at 4 weeks and 85%–93% at 8 weeks of treatment. The rapamycin plus prednisolone treatment resulted in significant decreases in the frequency of B cells, plasma cells, and IgG-secreting and AAV9-specific Ab-producing plasma cells in bone marrow. The rapamycin plus prednisolone treatment also significantly reduced frequencies of IgD−IgG+ class-switched/FAS+CL7+ germinal center B cells, and of activated CD4+ T cells expressing PD1 and GL7, in spleen. These data suggest that rapamycin plus prednisolone has selective inhibitory effects on both T helper type 2 support of B cell activation in spleen and on bone marrow plasma cell survival, leading to effective AAV9 Abs depletion. This promising immunomodulation approach is highly translatable, and it poses minimal risk in the context of therapeutic benefits promised by gene therapy for severe monogenetic diseases, with a single or possibly a few treatments over a lifetime.

Inactivation of CK1α in multiple myeloma empowers drug cytotoxicity by affecting AKT and β-catenin survival signaling pathways.

Recent evidence indicates that protein kinase CK1α may support the growth of multiple myeloma (MM) plasma cells. Here, by analyzing a large cohort of MM cases, we found that high CK1α mRNA levels are virtually associated with all MM patients. Moreover, we provided functional evidence that CK1α activity is essential for malignant plasma cell survival even in the protective niche generated by co-cultures with bone marrow stromal cells. We demonstrated that CK1α inactivation, while toxic for myeloma cells, is dispensable for the survival of healthy B lymphocytes and stromal cells. Disruption of CK1α function in myeloma cells resulted in decreased Mdm2, increased p53 and p21 and reduced expression of β-catenin and AKT. These effects were mediated partially by p53 and caspase activity. Finally, we discovered that CK1α inactivation enhanced the cytotoxic effect of both bortezomib and lenalidomide. Overall, our study supports a role for CK1α as a potential therapeutic target in MM in combination with proteasome inhibitors and/or immunomodulatory drugs.

"Inflamm-aging" influences immune cell survival factors in human bone marrow.

The bone marrow (BM) plays a key role in the long‐term maintenance of immunological memory. However, the impact of aging on the production of survival factors for effector/memory T cells and plasma cells in the human BM has not been studied. We now show that the expression of molecules involved in the maintenance of immunological memory in the human BM changes with age. While IL‐15, which protects potentially harmful CD8+CD28− senescent T cells, increases, IL‐7 decreases. IL‐6, which may synergize with IL‐15, is also overexpressed. In contrast, a proliferation‐inducing ligand, a plasma cell survival factor, is reduced. IFN‐y, TNF, and ROS accumulate in the BM in old age. IL‐15 and IL‐6 expression are stimulated by IFN‐y and correlate with ROS levels in BM mononuclear cells. Both cytokines are reduced by incubation with the ROS scavengers N‐acetylcysteine and vitamin C. IL‐15 and IL‐6 are also overexpressed in the BM of superoxide dismutase 1 knockout mice compared to their WT counterparts. In summary, our results demonstrate the role of inflammation and oxidative stress in age‐related changes of immune cell survival factors in the BM, suggesting that antioxidants may be beneficial in counteracting immunosenescence by improving immunological memory in old age.

IL-10 production in murine IgM+ CD138hi cells is driven by Blimp-1 and downregulated in class-switched cells.

In contrast to antibody-induced inflammatory responses, some B-cell subpopulations suppress inflammation through the production of interleukin (IL)-10. However, the mechanisms underlying Il10 gene expression during B-cell development is elusive. Here, we identify IgM+ B220lo CD138hi cells responsible for marked IL-10 production in the bone marrow and spleen of mice. These murine IL-10-producing cells predominantly secrete IgM and have unique characteristics of long-lived plasma cells in spite of high expression of surface IgM. We found that IL-10 production is strongly correlated with the expression level of Prdm1 (encoding the Blimp-1 protein), an essential regulator of plasma cell development. Furthermore, overexpression of Prdm1 induces Il10 expression in naïve B cells. Immunoglobulin class-switching recombination events resulted in the downregulation of both Il10 and Prdm1 expression in differentiating B cells. Thus, the prolonged elevation of Blimp-1 expression during the formation of IgM+ CD138hi cells without class-switching elicits IL-10 production. Adoptive transfer of Il10-deficient B cells into B-cell-deficient mice demonstrated that IgM+ CD138hi cell-derived IL-10 supports the survival of class-switched plasma cells and their antibody production in response to antigen challenge. These findings reveal an important role for IL-10 secretion by IgM+ CD138hi cells in the complete and efficient humoral response.

Tyrosine kinase c-Abl regulates the survival of plasma cells

Tyrosine kinase c-Abl plays an important role in early B cell development. Its deletion leads to reduced pro- and pre-B cell generation in mice. However, its function in B cell terminal differentiation remains unexplored. Here, we used c-Ablf/f Aicdacre/+ mice, in which c-Abl is ablated only in antigen-activated B cells, to study the role of c-Abl in germinal center (GC) B and antibody-secreting plasma cell formation. Upon challenge with a model antigen, we found normal GC and memory B but reduced plasma cells and antigen-specific antibody response in the mutant mice. In-vitro studies revealed that plasma cells lacking c-Abl could be generated but did not accumulate in culture, indicative of survival defect. They also exhibited impaired STAT3 phosphorylation. The plasma cell defects could be rectified by introduction of Bim-deficiency or delivery of colivelin, a STAT3 activator, into c-Ablf/f Aicdacre/+ mice. Hence, c-Abl signalling regulates the survival of plasma cells.

Regulation of eosinophil development and survival.

Eosinophils are a subset of granulocytes generally associated with type 2 immune responses. They can contribute to protection against helminths but also mediate pro-inflammatory functions during allergic immune responses. Only recently, eosinophils were also found to exert many other functions such as regulation of glucose and fat metabolism, thermogenesis, survival of plasma cells, and antitumor activity. The mechanisms that control eosinophil development and survival are only partially understood. Here we review new findings regarding the role of cell-extrinsic and cell-intrinsic factors for eosinophilopoiesis and eosinophil homeostasis. Several reports provide new insights in the regulation of eosinophil development by transcription factors, miRNAs and epigenetic modifications. Danger signals like lipopolysaccharide or alarmins can activate eosinophils but also prolong their lifespan. We further reflect on the observations that eosinophil development is tightly controlled by the unfolded protein stress response and formation of cytoplasmic granules. Eosinophils emerge as important regulators of diverse biological processes. Their differentiation and survival is tightly regulated by factors that are still poorly understood. Newly identified pathways involved in eosinophilopoiesis and eosinophil homeostasis may lead to development of new therapeutic options for treatment of eosinophil-associated diseases.

Human Basophils Modulate Plasma Cell Differentiation and Maturation

Basophils represent <1% of circulating leukocytes. They play a crucial role during allergy and helminth-induced Th2 responses. However, recent data also suggest a contribution to the pathogenesis of autoimmune diseases. Basophils from patients with systemic lupus erythematosus show an activated phenotype, correlating to disease activity. Furthermore, murine basophils or their mediators enhance memory responses and plasma cell (PC) survival, suggesting that they directly modulate the function of B cells. This is highly relevant with respect to human allergy and autoimmunity because a possible modulation of B cell differentiation by basophils could point to new therapeutic targets. Therefore, the interaction between human B cells and basophils and the mechanism underlying this interaction were investigated in detail. Using two different methods to induce PC differentiation, we found that human basophils enhance B cell proliferation, class switching, differentiation into PC, maturation of PC, and production of Igs, especially IgG. Basophil supernatants enhanced the expression of the B cell markers CD23 and CD40, which are important for B cell differentiation into IgG-producing PC. This was mainly IL-4 dependent. IL-3 amplified the number of PC in vitro, and acted synergistically with basophils in enhancing Ab production. Thus, human basophils modulate B cell differentiation into Ab-producing PC. Their contribution as modulators and effectors during allergy and autoimmunity should be considered when designing new therapeutic options.

Neutrophils releasing IL-17A into NETs are essential to plasma cell differentiation in inflamed tissue dependent on IL-1R

Interleukin (IL) 17A in chronic inflammation is also produced by innate immune cells as neutrophils. Mice with chronic humoral response induced by venom of Thalassophryne nattereri (VTn) proved to be a good tool for evaluating the impact of IL-17A on the development of long-lived plasma cells in the inflamed peritoneal cavity. Here, we report that VTn induces IL-17A production by neutrophils accumulating in the peritoneal cavity and triggers the extrusion of IL-17A along with neutrophil extracellular traps (NETs). Neutrophil depletion reduced the number of IL17A-producing cells in VTn-immunized mice and blocked the differentiation of long-lived plasma cells. Specific antibody production and survival of long-lived plasma cells was ablated in VTn-immunized mice deficient in CD4, while CD28 signaling had the opposite effect on differentiation of long-lived plasma cells. Further, maturation of long-lived plasma cells in inflamed peritoneal cavity was IL-1R1 and COX-2 dependent. Finally, when both the Raf-MEK-ERK pathway and the IL-17A or IL-1R1 activities were blocked, neutrophils were unable to promote the differentiation of memory B cells into long-lived plasma cells, confirming the essential role of neutrophils and IL-17A along with NETs in an IL-1/IL-1R-dependent manner as the novel helping partner for plasma cell differentiation in chronically inflamed tissues.

Urinary B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL): potential biomarkers of active lupus nephritis.

B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) help in B cell activation, maintenance and plasma cell survival. B cell infiltration has been demonstrated in kidneys of patients with lupus nephritis (LN). Serum levels of BAFF and APRIL have shown inconsistent relationships with lupus disease activity. We evaluated urinary levels of BAFF and APRIL as biomarker for LN. Thirty-six patients with proliferative lupus nephritis (AN), 10 with active lupus without nephritis (AL) and 15 healthy controls (HC) were studied. APRIL and BAFF levels were measured in both serum and urine using enzyme-linked immunosorbent assay (ELISA). Urine levels were normalized for urinary creatinine excretion. Urine levels were correlated with conventional disease activity markers and histology. Levels were reassessed in 20 AN patients at 6 months after treatment with cyclophosphamide. Urinary APRIL (uAPRIL) and BAFF (uBAFF) levels were raised significantly in AN. uAPRIL, but not uBAFF, correlated moderately with renal Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) in AN (r = 0·36, P < 0·05). On receiver operator curve (ROC) analysis, uBAFF and uAPRIL showed an area under the curve (AUC) of 0·825 and 0·781, respectively, in differentiating between nephritis and non-nephritis, which performed better than low C3, C4 and raised anti-dsDNA antibodies. There was no correlation of serum levels with uBAFF (r = 0·187, P = 0·261) and uAPRIL (r = 0·114, P = 0·494). uAPRIL levels reduced after treatment (mean 125 pg/mg to 36 pg/mg, P < 0·05). uBAFF levels reduced in 16 responders while two of four non-responders had increase in levels. Thus, uBAFF and uAPRIL are potential biomarkers of proliferative lupus nephritis.

Toll-Like Receptor 9 Stimulation Induces Aberrant Expression of a Proliferation-Inducing Ligand by Tonsillar Germinal Center B Cells in IgA Nephropathy.

The TNF family member a proliferation-inducing ligand (APRIL; also known as TNFSF13), produced by myeloid cells, participates in the generation and survival of antibody-producing plasma cells. We studied the potential role of APRIL in the pathogenesis of IgA nephropathy (IgAN). We found that a significant proportion of germinal centers (GCs) in tonsils of patients with IgAN contained cells aberrantly producing APRIL, contributing to an overall upregulation of tonsillar APRIL expression compared with that in tonsils of control patients with tonsillitis. In IgAN GC, antigen-experienced IgD-CD38+/-CD19+ B cells expressing a switched IgG/IgA B cell receptor produced APRIL. Notably, these GC B cells expressed mRNA encoding the common cleavable APRIL-α but also, the less frequent APRIL-δ/ζ mRNA, which encodes a protein that lacks a furin cleavage site and is, thus, the uncleavable membrane-bound form. Significant correlation between TLR9 and APRIL expression levels existed in tonsils from patients with IgAN. In vitro, repeated TLR9 stimulation induced APRIL expression in tonsillar B cells from control patients with tonsillitis. Clinically, aberrant APRIL expression in tonsillar GC correlated with greater proteinuria, and patients with IgAN and aberrant APRIL overexpression in tonsillar GC responded well to tonsillectomy, with parallel decreases in serum levels of galactose-deficient IgA1. Taken together, our data indicate that antibody disorders in IgAN associate with TLR9-induced aberrant expression of APRIL in tonsillar GC B cells.

BAFF and CD4 T-Cells Are Major Survival Factors for Splenic Plasma Cells in B Cell Depletion Context: Implications for Autoimmune Diseases

The use of monoclonal anti-CD20 antibody (Rituximab) has greatly improved the treatment of B-cell mediated autoimmune diseases, albeit with variable outcomes. Our previous data in humans suggested that Rituximab induced paradoxically the settlement of splenic long-lived plasma cells (LLPC) in the context of 2 autoimmune cytopenia, immune thrombocytopenia and warm autoimmune hemolytic anemia (1) (2). The presence of splenic autoreactive LLPC explained the failure of Rituximab treatment. To investigate whether this mechanism could have a general relevance and decipher the cellular and molecular mechanism of this process, we used both non auto-immune and auto-immune mouse models.We have taken advantage of the knock-in transgenic mouse model AID-CreERT2-EYFP, which allows the irreversible expression of EYFP in B cells engaged in a germinal center-dependent immune response after tamoxifen regimen, to follow plasma cells (PC) at different times of immunization by sheep red blood cells, and upon anti-CD20 regimen (clone 18B12, Biogen Idec), in the spleen and bone marrow (3).By using a set of diagnostic genes that allowed us to distinguish short-lived and long-lived plasma cells, we compared the transcriptional program by multiplex PCR of EYFP+ B220- PC from controls and anti-CD20 treated mice, immunized and analyzed at the same time, corresponding to the nadir of B-cell depletion. While splenic PC of untreated mice displayed an intermediate profile between short-lived and long-lived plasma cells, splenic PC from anti-CD20 treated mice composed a homogeneous population that displayed a more mature program, similar to the one of natural long-lived bone marrow PC. The absolute number of splenic EYFP+ B220- did not change upon anti-CD20 treatment indicating that B-cell depletion promoted PC differentiation rather than a long-lived PC selection. We identified BAFF (B-cell activating factor) as a major player of this process. Indeed, as described in human spleens, we observed that BAFF level was increased in the supernatants of splenocytes after B-cell depletion. Above all, combination of anti-CD20 and anti-BAFF (clone 10F4, GSK) antibodies dramatically reduced the number of splenic EYFP+B220- LLPC (decrease >5 fold compared with anti-CD20 and control groups, P < 0.001). Targeting BAFF had no major impact on protective long-lived bone marrow PC as IgG1 level in the sera remained unchanged after combination therapy. We identified neutrophils as the main source of BAFF production in the spleen. Finally, CD4+ T-cells also appeared to play a key role in context of B-cell depletion for supporting plasma cell survival in the spleen as they appeared to closely interact with EYFP+ plasma cells by confocal microscopy. Moreover, their depletion (clones YTS 191.1 or GK 1.5, Bioxcell) in vivo induced a significant decrease in the number of splenic LLPCs (decrease > 2 fold compared with anti-CD20 group, P < 0.05). To assess whether B-cell depletion could also modify the splenic plasma cell program in an auto-immune context characterized by an ongoing immune response, we used NZB/NZW mice that spontaneously develop a disease closely resembling human systemic lupus. In line with our previous findings, anti-CD20 treatment also promoted the differentiation of LLPC in the spleen of the NZB/NZW model, while a treatment combining anti-CD20 with anti-BAFF induced a marked reduction in total PC numbers(decrease > 3 fold compared with anti-CD20 group, P < 0.05).In conclusion, the process of PC maturation upon anti-CD20 treatment appeared to be a general mechanism, both in non auto-immune and auto-immune models. We identified BAFF and CD4+ T-cells as key factors in the splenic environment responsible for the emergence of such LLPC. Finally, our results suggest that interfering with the plasma cell survival niche with monoclonal anti-BAFF antibody at the time of B-cell depletion might greatly improve the response rate in B-cell mediated auto-immune cytopenia.(1) Mahevas M, et al, Journal of Clinical Investigation , 2013(2) Mahevas M, et al, Journal of Autoimmunity, 2015(3) Dogan I, et al, Nature Immunology, 2009 DisclosuresNo relevant conflicts of interest to declare.

An APRIL Based Chimeric Antigen Receptor to Simultaneously Target BCMA and TACI in Multiple Myeloma (MM) Has Potent Activity in Vitro and in Vivo

Immune based strategies have shown early promise in multiple myeloma (MM), and are likely to prove a valuable addition to the therapeutic platforms for these patients. B cell maturation antigen (BCMA) is a promising therapeutic target with selective expression on plasma cells, including MM cells. In the first report of BCMA targeting therapies, an anti-BCMA scFv-based chimeric antigen receptor (CAR) had clinical activity, albeit with short lived responses (Ali et al, 2016). Observed challenges included low expression level of BCMA, shedding of BCMA and potential loss of expression.In addition to BCMA, MM cells also express transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) which functions in plasma cell survival and differentiation. TACI is selectively expressed within lymphoid tissue and at reduced levels compared to BCMA, as measured by RT-PCR on normal tissues. TACI and BCMA share a common ligand called a proliferating ligand (APRIL), a compact self-protein forming a trimer and binding with nanomolar affinity to TACI and BCMA. The natural trimerization of APRIL would serve to enhance cell binding, especially important in the case of low levels of target antigen. Concurrent targeting of two tumour antigens, by increasing antigen density, would improve efficacy as well as reducing the risk of antigen negative disease escape.We quantified APRIL targets on primary MM cells by flow cytometry and observed BCMA expression in all of 41 patients tested (median 1051, range 105-8323 antibodies bound per cell, ABC) with most patients (78%) also expressing TACI (333, 0-21301 ABC). We designed an APRIL based 3rd generation chimeric antigen receptor (APRIL-CAR) comprising an engineered APRIL protein as antigen-binding domain. Peripheral blood mononuclear cells (PBMCs) were activated, transduced with APRIL-CAR and CD56-depleted before testing against cell lines expressing a wide range of BCMA and TACI (1005-1.5x10^5 and 2200-1.6x10^5 ABC respectively). We confirmed the cytolytic capability of APRIL-CAR against engineered targets, even at the low antigen levels observed in primary cells. Using standard chromium release, there was significant cytolysis of the lowest antigen expressers, with 40.9±3.8% (mean±SEM) and 42.6±5.7% cell death in SUPT1BCMAlo and SUPT1TACIlo respectively cf 15.0±5.8% for control SUPT1NT targets, at an E:T ratio of 4:1 (n=4, p<0.01 and p<0.05). When we lowered the E:T ratio, APRIL-CAR continued to show potent activity, with cytolysis of SUPT1BCMAlo/ SUPT1TACIlo targets at 1:10 (71.7±3.8% and 70.4±6.3% cell death respectively cf 21.6±5.8% for SUPT1NT, n=4, p<0.001 for both) and human myeloma cell lines at 1:32 (MM.1s and U266, 56.2±3.9% and 25.1±3.9% target cytolysis by APRIL-CAR and 4.7±11.6% and 6.6±3.9% by PBMCs NT respectively, n=5, p<0.01 for both).We confirmed cytolytic activity of APRIL-CAR against primary MM cells. Allogeneic PBMCs transduced with APRIL-CAR and CD56-depleted were co-cultured 1:1 with CD138+ bone marrow MM cells from 5 patients. Despite variable BCMA and TACI expression between patient samples (BCMA 1224-7728 and TACI 563-1213 ABC), tumour cytolysis was evident in all cases (APRIL-CAR 72.9±12.2% specific cell death cf media control 2.8±15.3%; p<0.05).Finally we tested the in vivo activity of APRIL-CAR in an intramedullary model established with tail vein injection of MM.1s.Fluc cells into NOD scid gamma (NSG) mice. Animals were analysed by bioluminescent imaging and FACS analysis of bone marrow for tumour cells. Complete clearance of established intramedullary disease was seen 5 days after infusion of 5x10^6 APRIL-CAR positive PBMCs in 12/12 animals. Elimination of tumour cells was confirmed in the bone marrow (p<0.01) with concurrent detection of APRIL-CAR T cells by FACS analysis.In summary we report for the first time a 3rd generation CAR utilising a natural ligand to simultaneously target two antigens found on MM cells. Our APRIL-based CAR demonstrated potent activity at the low levels of BCMA and TACI found in primary MM cells, at low (physiological) E:T ratios, and in an intramedullary MM model. This APRIL based CAR holds particular promise and deserves testing in clinical studies. DisclosuresLee:Bloodwise: Research Funding; Autolus Ltd: Patents & Royalties: APRIL based chimeric antigen receptor. Draper:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based CAR. Chaplin:Autolus Ltd: Equity Ownership. Philip:Autolus: Equity Ownership. Thomas:Autolus Ltd: Employment. Kokalaki:Autolus Ltd: Employment. Francis:Autolus Ltd: Employment. Yong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

Myeloma Exosomes Prime the Microenvironment to Support Survival and Growth of Myeloma Cells

IntroductionMyeloma bone marrow serum primes healthy donor (HD) bone marrow cells to support the survival and growth of myeloma plasma cells (MMPC) in co-culture. Following interaction with the MMPC, the phenotype of the HD bone marrow stroma changes and the expression of immune regulatory factors is increased while expression of genes associated with osteoblastogenesis and WNT signaling is suppressed. The aim of this study was to determine whether exosomes in the myeloma bone marrow serum have a role in priming the microenvironment.MethodsSerum was separated from clotted bone marrow aspirates from myeloma patients obtained during scheduled clinic visits, and from healthy donors of similar ages.Exosomes were prepared from 1-2 ml of serum using Invitrogen’s Total Exosome Isolation kit according to manufacturer’s instruction. Exosomes were 30-130 nm in diameter as determined by Dynamic Light Scatter on a Brookhaven Instruments Corp. ZetaPlus Particle Sizing Software, and visualized by scanning electron microscopy. Small RNA (≤200 nucleotides) was isolated using Qiagen’s miRNeasy kits, quality and quantity assessed with Qubit 2. 96 microRNAs (miRs) were analyzed by qRT-PCR on Fluidigm’s BioMark platform using 96x96 multiplex chips and normalized to spiked in C. elegance miR mimetic.Primary CD138-expressing cells were isolated from EDTA-anticoagulated bone marrow aspirates. Luciferase-expressing stroma-dependent myeloma cells were described previously (Bam, BMC Cancer 2015:864). Healthy donor mesenchymal stem cells (MSC) were prepared from bone particles obtained during orthopedic surgery, and cultured in a-MEM supplemented with 10% FBS.Primary myeloma cells or luciferase-expressing stroma dependent myeloma cells were added to wells containing monolayers of MSC in α-MEM supplemented with 10% FBS in 96 well culture plates. For each experiment 3 conditions were used: A control group, a group to which 10% myeloma bone marrow serum was added, and a third group to which exosomes isolated from a matching bone marrow serum aliquot were added. The number of primary MMPC dictated that these experiments were carried out in duplicates, while the stroma dependent cells were plated in 5 replicates. Viable primary cells counts or luciferase bioluminescence were evaluated after 4 days of co-culture.ResultsExosomes supported survival and growth of primary MMPC in co-culture with HD MSC, but had no effect on MMPC cultured alone, indicating that the myeloma bone marrow serum exosomes prime the MSC to support the MMPC. Exosomes from 5 of the 7 patients supported growth of stroma-dependent myeloma cells co-cultured with HD MSC. In a representative experiment, the bioluminescence of stroma-dependent myeloma cells was 46% higher in co-cultures supplemented with bone marrow serum (p=7.3E-6) and 23% higher (p=2.0E-6) when exosomes were used. The effect was reproduced when primary MMPC were used: in a representative experiment, the number of primary MPC recovered from the co-cultures supplemented with bone marrow serum was 41% higher, and when supplemented with exosomes 28% higher than in controls. Primary MMPC did not survive in the absence of MSCs and bone marrow serum or exosomes did not enhance their survival.ConclusionsExosome from bone marrow serum of myeloma patients prime the bone marrow microenvironment to support survival and growth of primary MMPC. Bone marrow serum is more effective, indicating that other factors in the serum are also important. The differences in microRNA contents between healthy donor and myeloma exosomes may provide an insight of the regulatory mechanisms molding the myeloma bone marrow microenvironment. DisclosuresMorgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.

First in Human Study with GSK2857916, an Antibody Drug Conjugated to Microtubule-Disrupting Agent Directed Against B-Cell Maturation Antigen (BCMA) in Patients with Relapsed/Refractory Multiple Myeloma (MM): Results from Study BMA117159 Part 1 Dose Escalation

BackgroundExpression of BCMA, a cell surface receptor in the TNF superfamily, is restricted to B cells at later stages of differentiation and is requisite for the survival of long lived plasma cells. BCMA is expressed on MM cells at variable levels.GSK2857916 is a humanized IgG1 anti-BCMA antibody conjugated to the microtubule disrupting agent monomethyl auristatin-F via a stable, protease-resistant maleimidocaproyl linker. Upon binding to BCMA, GSK2857916 is rapidly internalized and active drug is released within the cell. GSK2857916 also exhibits enhanced antibody-dependent cell-mediated cytotoxicity resulting from afucosylation of the FC domain which increases affinity to FCγRIIIa expressed on immune effector cells. The rationale for investigating GSK2857916 in MM is supported by the restricted pattern of BCMA expression and by evidence from preclinical studies.MethodsBMA117159 (NCT02064387) is a Phase I open-label study investigating safety, pharmacokinetics (PK), pharmacodynamics (PD), antidrug antibodies (ADA) and clinical activity of GSK2857916 in relapsed/refractory MM and other hematologic malignancies expressing BCMA. Dose escalation (Part 1) is conducted exclusively in MM patients; primary objective is safety, determination of maximum tolerated dose (MTD) and recommended phase 2 dose. GSK2857916 is administered every 3 weeks as a1 hr intravenous infusion with no mandatory prophylaxis for infusion related reactions (IRR). Bayesian Logistic Regression Model is applied for dose recommendations. Eligible MM patients must have prior treatments with alkylators, proteasome inhibitors, immunomodulators and stem cell transplantation, if transplant eligible, and have documented disease progression on or within 60 days of last therapy. Additional key inclusion criteria include: ANC >1x109/L; hemoglobin >8 g/dL; PLT >50x109/L; INR <1.5; PTT <1.5xULN; total bilirubin ≤1.25xULN; AST and ALT <1.5xULN; serum creatinine <1.2xULN or calculated creatinine clearance >60 mL/min; albuminuria <500mg/24hr; LVEF >50% and troponin <1xULN. Patients remain on treatment until progression, unacceptable toxicity, consent withdrawal or completing 16 treatment cycles. Blood is collected for PK and ADA analyses; blood and bone marrow aspirates are collected for PD analyses.ResultsTo date 24 MM patients have enrolled and completed the 21 day DLT observation period. Patients were enrolled at the following 8 dose levels: 0.03 (n=1); 0.06 (n=1); 0.12 (n=4); 0.24 (n=4); 0.48 (n=4); 0.96 (n=3); 1.92 (n=4), and 3.4 mg/kg (n=3). The median age is 60 years (range 39-71); 50% are male. Eighty-three percent received ≥4 prior lines. Sixty-three percent had IgG and 29% had IgA types. Seven patients (29%) had adverse cytogenetics defined as del17p13, or t(4;14) . Overall, 23 patients (96%) experienced adverse events (AE); the most frequent (≥20%) regardless of cause were nausea (42%), fatigue (38%), anemia (29%), chills (29%), pyrexia (29%), thrombocytopenia (29%), dry eye (21%), and hypercalcemia (21%). Grade 3/4 AEs reported in ≥10% of patients were thrombocytopenia, anemia, and neutropenia. Eight serious AEs were reported in 6 patients with 1 event of limbal stem cell dysfunction considered drug related; this event has not resolved. There were no AEs leading to treatment discontinuation. Four patients required dose reduction due to AEs: ocular toxicity (n=1), corneal disorder/ocular toxicity (n=1), dry eyes (n=1), and keratitis (n=1). IRRs (all Grade 1 or 2) were reported in 7 patients (29%) across dose levels and occurred with the first dose administration. The most frequent symptoms were chills. No DLTs were reported. Sixteen patients discontinued treatment due to disease progression (n=14), patient decision (n=1), completing 16 cycles of scheduled treatment (n=1); 8 patients are ongoing. There was1 MR at 0.24 mg/kg, and 1 VGPR, 3 PR, and1 MR at doses ≥0.96 mg/kg; resulting in a clinical benefit rate of 25% (unconfirmed responses).ConclusionGSK2857916 was well tolerated with no DLTs up to 3.4mg/kg q3w; MTD was not reached. AEs were manageable with ocular toxicity emerging as the most frequent reason for dose modifications. Clinical activity has been seen at higher doses. Complete safety, ADA, PD, and clinical activity data from all dose levels evaluated in Part 1 for q3w schedule will be presented.Study is funded by GlaxoSmithKline; drug linker technology is licensed from Seattle Genetics DisclosuresCohen:Janssen: Consultancy; Bristol-Meyers Squibb: Consultancy, Research Funding. Trudel:Glaxo Smith Kline: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Equity Ownership, Honoraria; Oncoethix: Research Funding; BMS: Honoraria; Amgen: Honoraria. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees. Anderson:Takeda: Speakers Bureau; AMGEN/Onyx: Speakers Bureau; Celegene: Speakers Bureau. DeWall:GlaxoSmithKline: Employment, Equity Ownership. Ellis:GlaxoSmithKline: Employment, Equity Ownership. He:GlaxoSmithKline: Employment, Equity Ownership. Mazumdar:GlaxoSmithKline: Employment, Equity Ownership. Wang:GlaxoSmithKline: Employment, Equity Ownership. Opalinska:GlaxoSmithKline: Employment, Equity Ownership. Voorhees:GlaxoSmithKline: Research Funding; Celgene, BMS: Honoraria; Takeda: Consultancy, Honoraria; Novartis: Consultancy.