Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells.

While a number of extrinsic factors are known to promote the survival of plasma cells (PCs), the signaling intermediates involved remain poorly characterized. Here we identified inducible nitric oxide synthase (iNOS) as an intermediate that supported the survival of PCs. PCs deficient in iNOS (Nos2(-/-) PCs) showed enhanced death in vitro, after transfer into congenic adoptive hosts, and in chimeras made with wild-type and Nos2(-/-) bone marrow. The iNOS-mediated protection involved activation of protein kinase G and modulation of endoplasmic reticulum stress components. Activation of caspases was also diminished. We found that iNOS was required for PCs to respond to some prosurvival mediators associated with bone marrow stromal cells and that at least one mediator, interleukin 6, fed directly into this pathway by inducing iNOS.

A novel PDK1 inhibitor, JX06, inhibits glycolysis and induces apoptosis in multiple myeloma cells.

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 µM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.

Mucosal Inducible NO Synthase-Producing IgA+ Plasma Cells in Helicobacter pylori-Infected Patients.

The mucosal immune system is relevant for homeostasis, immunity, and also pathological conditions in the gastrointestinal tract. Inducible NO synthase (iNOS)-dependent production of NO is one of the factors linked to both antimicrobial immunity and pathological conditions. Upregulation of iNOS has been observed in human Helicobacter pylori infection, but the cellular sources of iNOS are ill defined. Key differences in regulation of iNOS expression impair the translation from mouse models to human medicine. To characterize mucosal iNOS-producing leukocytes, biopsy specimens from H. pylori-infected patients, controls, and participants of a vaccination trial were analyzed by immunohistochemistry, along with flow cytometric analyses of lymphocytes for iNOS expression and activity. We newly identified mucosal IgA-producing plasma cells (PCs) as one major iNOS(+) cell population in H. pylori-infected patients and confirmed intracellular NO production. Because we did not detect iNOS(+) PCs in three distinct infectious diseases, this is not a general feature of mucosal PCs under conditions of infection. Furthermore, numbers of mucosal iNOS(+) PCs were elevated in individuals who had cleared experimental H. pylori infection compared with those who had not. Thus, IgA(+) PCs expressing iNOS are described for the first time, to our knowledge, in humans. iNOS(+) PCs are induced in the course of human H. pylori infection, and their abundance seems to correlate with the clinical course of the infection.

Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B-cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short-lived plasma cells, long-lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock-out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre- and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long-lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage.

Activity of aldehyde dehydrogenase in B-cell and plasma cell subsets of monoclonal gammopathy patients and healthy donors.

BACKGROUND: Aldehyde dehydrogenase (ALDH) is highly active in physiological stem cells as well as in tumor-initiating cells of some malignancies including multiple myeloma (MM). Finding higher activity of ALDH in some cell subsets in monoclonal gammopathies (MG) could identify potential source of myeloma-initiating cells (MICs). METHODS: Bone marrow of 12 MM, 9 monoclonal gammopathy of undetermined significance (MGUS), and 10 healthy donors (HD) were analyzed by flow cytometry. ALDH activity of B-cells and plasma cells (PC) was analyzed using Aldefluor. RESULTS: Similar changes of ALDH activity were found during B-cell development in HD and MG. Decreasing of ALDH activity from immature to naïve B-cells was found. In postgerminal stages, the activity started to increase, and in PCs, the ALDH activity was the same as in immature B-cells. Increased ALDH activity of all PC subsets compared to naïve B-cells was found in MM as well as in HD, while in MGUS, only CD19- PCs have higher ALDH activity. In HD, ALDH activity was higher in CD19+ PCs compared with MG. CONCLUSIONS: Our results indicate that changes of ALDH activity are the natural phenomenon in B-cell development; thus, high ALDH activity as a single marker is not appropriate for MICs identification.

Plasma cell alloantigen ENPP1 is expressed by a subset of human B cells with potential regulatory functions.

Plasma cell alloantigen 1 (PC1), also known as ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), is an enzyme involved primarily in hydrolysis of adenosine triphosphate at the cell surface. Although the expression pattern of PC1 is relatively broad, its expression in B cells is found at significant levels only in terminally differentiated germinal center B cells, plasma cells and a subset of B-1a cells in mice. Here we describe studies designed to determine whether expression of PC1 might define novel populations of human B cells with similarities to mouse B cells. We found that PC1 is expressed in small populations of human B lineage cells in peripheral blood, cord blood, tonsils, bone marrow and pediatric peritoneal fluid, with the highest levels in plasma cells. The characteristics of human PC1(+) B cells differ from mouse peritoneal B-1a subsets and from features of the human CD20(+)CD27(+)CD43(+)CD70(-) B-cell subset proposed to be human B-1 cells. Expression of PC1 was greatly increased in B cells stimulated with the combination of CD40 ligand, interleukin (IL)-4 and IL-21. In addition, PC1(+) B cells activated CD4(+) T regulatory cells. ENPP1 thus defines a subset of human B cells that differs significantly from mouse peritoneal B-1a and proposed human B-1 cells.

Modeling multiple myeloma by AID-dependent conditional activation of MYC.

Efforts to create a mouse model that provides even a phenocopy of human multiple myeloma (MM) have been unsuccessful. In this issue of Cancer Cell, Bergsagel and colleagues describe an apparent solution to this problem by creating a model in which a MYC transgene containing a stop codon and flanking Ig kappa regulatory sequences is activated sporadically in germinal center B cells by AID-dependent somatic hypermutation that reverts the stop codon. Although much remains to be done to fully characterize this model, this approach is likely to impact the creation of sporadic models for other kinds of germinal center B cell tumors.

AID-dependent activation of a MYC transgene induces multiple myeloma in a conditional mouse model of post-germinal center malignancies.

By misdirecting the activity of Activation-Induced Deaminase (AID) to a conditional MYC transgene, we have achieved sporadic, AID-dependent MYC activation in germinal center B cells of Vk*MYC mice. Whereas control C57BL/6 mice develop benign monoclonal gammopathy with age, all Vk*MYC mice progress to an indolent multiple myeloma associated with the biological and clinical features highly characteristic of the human disease. Furthermore, antigen-dependent myeloma could be induced by immunization with a T-dependent antigen. Consistent with these findings in mice, more frequent MYC rearrangements, elevated levels of MYC mRNA, and MYC target genes distinguish human patients with multiple myeloma from individuals with monoclonal gammopathy, implicating a causal role for MYC in the progression of monoclonal gammopathy to multiple myeloma.

Human plasma cells express granzyme B.

While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

IL-21 and CD40L synergistically promote plasma cell differentiation through upregulation of Blimp-1 in human B cells.

After undergoing Ig somatic hypermutation and Ag selection, germinal center (GC) B cells terminally differentiate into either memory or plasma cells (PCs). It is known that the CD40L and IL-21/STAT3 signaling pathways play critical roles in this process, yet it is unclear how the B cell transcription program interprets and integrates these two types of T cell-derived signals. In this study, we characterized the role of STAT3 in the GC-associated PC differentiation using purified human tonsillar GC B cells and a GC B cell-like cell line. When primary GC B cells were cultured under PC differentiation condition, STAT3 inhibition by AG490 prevented the transition from GC centrocytes to preplasmablast, suggesting that STAT3 is required for the initiation of PC development. In a GC B cell-like human B cell line, although IL-21 alone can induce low-level Blimp-1 expression, maximum Blimp-1 upregulation and optimal PC differentiation required both IL-21 and CD40L. CD40L, although having no effect on Blimp-1 as a single agent, greatly augmented the amplitude and duration of IL-21-triggered Jak-STAT3 signaling. In the human PRDM1 locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation.

IL-2 requirement for human plasma cell generation: coupling differentiation and proliferation by enhancing MAPK-ERK signaling.

Mature B cell differentiation involves a well-established transcription factor cascade. However, the temporal dynamics of cell signaling pathways regulating transcription factor network and coordinating cell proliferation and differentiation remain poorly defined. To gain insight into the molecular processes and extrinsic cues required for B cell differentiation, we set up a controlled primary culture system to differentiate human naive B cells into plasma cells (PCs). We identified T cell-produced IL-2 to be critically involved in ERK1/2-triggered PC differentiation. IL-2 drove activated B cell differentiation toward PC independently of its proliferation and survival functions. Indeed, IL-2 potentiated ERK activation and subsequent BACH2 and IRF8 downregulation, sustaining BLIMP1 expression, the master regulator for PC differentiation. Inhibition of the MAPK-ERK pathway, unlike STAT5 signaling, impaired IL-2-induced PC differentiation and rescued the expression profile of BACH2 and IRF8. These results identify IL-2 as a crucial early input in mature B cell fate commitment.

Natural pesticide dihydrorotenone arrests human plasma cancer cells at the G0/G1 phase of the cell cycle.

Dihydrorotenone (DHR) is a natural pesticide used for farming including organic produces. We recently found that DHR induces human plasma cell apoptosis by provoking endoplasmic reticulum stress. In the present study, we found that DHR arrested human plasma cancer cells at the G0/G1 phase of the cell cycle. Mechanistical studies demonstrated that cell cycle arrest was associated with downregulated cell cycle promotors including cyclin D2, cyclin D3, cyclin-dependent kinases (CDK4, CKD6), and phosphorylated-Rb. DHR inhibited cyclin D2 transactivation, thus inhibiting its mRNA expression. In addition, DHR upregulated the cell cycle repressors p21 and p53. DHR also increased the phosphorylation level of p53, suggesting the upregulated transactivation function of p53, which was confirmed by the induction of p21, a substrate of activated p53. Moreover, DHR downregulated AKT and ERK phosphorylation, an incentive of cell cycle progression. Therefore, these results collectively demonstrated that DHR disrupts the cell cycle progress, which suggests that DHR is toxic to human plasma cells. Caution is thus suggested when handling with this agent.

[Immune status estimation algorithm in irritable bowel syndrome patients with food intolerance].

OBJECTIVE: To develop an algorithm for evaluation of the immune status in IBS patients with food intolerance. MATERIALS AND METHODS: 42 patients with IBS were observed. The diagnosis is based Rome III criteria. Determination of the concentration of serum allergen-IgG-antibodies to food allergens, IgG, IgA, IgM, IgE and immunoregulatory substances IL-5, TGF ß1, IL-10, IL-4 IL-2, IL-13 were determined by enzyme immunoassay. All the patients received rotational diet therapy based on immunoassay data in addition to medical treatment of the basis disease. The control group comprised 15 healthy individuals. RESULTS: Food intolerances basically to foods proteinaceous was diagnosed in 25 patients with IBS (59.5% of cases). The effectiveness of diet therapy was 68% (17 patients). Analysis of clinical and laboratory data revealed that the implementation of food intolerance in patients with IBS reducing the levels of IL-10 and TGFß1. An effective rotational diet of food intolerance in IBS patients had significantly (p < 0.05) increase IgA levels. CONCLUSION: Food intolerance in IBS patients characterized an increased synthesis of allergen-specific IgG antibodies, insufficient production of anti-inflammatory immunoregulatory substances IL-10 and TGFß1, and intact cytokines IL-2, - 4, - 5, - 13, IgG and IgM in serum. Efficiency rotary diet in patients with IBS is accompanied by positive clinical dynamics, increase levels of anti-inflammatory substances and serum IgA.

Disruption of heparan sulfate proteoglycan conformation perturbs B-cell maturation and APRIL-mediated plasma cell survival.

The development and antigen-dependent differentiation of B lymphocytes are orchestrated by an array of growth factors, cytokines, and chemokines that require tight spatiotemporal regulation. Heparan sulfate proteoglycans specifically bind and regulate the bioavailability of soluble protein ligands, but their role in the immune system has remained largely unexplored. Modification of heparan sulfate by glucuronyl C5-epimerase (Glce) controls heparan sulfate-chain flexibility and thereby affects ligand binding. Here we show that Glce deficiency impairs B-cell maturation, resulting in decreased plasma cell numbers and immunoglobulin levels. We demonstrate that C5-epimerase modification of heparan sulfate is critical for binding of a proliferation inducing ligand (APRIL) and that Glce-deficient plasma cells fail to respond to APRIL-mediated survival signals. Our results identify heparan sulfate proteoglycans as novel players in B-cell maturation and differentiation and suggest that heparan sulfate conformation is crucial for recruitment of factors that control plasma cell survival.

Osteoprotegerin is associated with aneurysm diameter and proteolysis in abdominal aortic aneurysm disease.

OBJECTIVE: Serum osteoprotegerin (OPG) concentrations have previously been associated with growth of abdominal aortic aneurysms (AAAs). In vitro experiments showed that OPG promotes matrix metalloprotease (MMP) release from monocytes and vascular smooth muscle cells. We hypothesized that OPG expression is increased in human AAAs and is associated with proteolysis. METHODS AND RESULTS: AAA biopsies were collected from 329 patients. We assessed the concentrations of OPG, cathepsins A, B, and S as well as the activity of MMP-2 and MMP-9. The AAA wall infiltration by macrophages, lymphocytes, and plasma cells was estimated by immunohistochemistry. The concentration of OPG correlated positively with aortic diameter (<55 mm: 16.1 [5.8-28.7], 55-70 mm: 21.9 [10.2-36.0], >70 mm: 24.0 [13.5-52.9] ng OPG/mg total amount of protein, P=0.020), cathepsin A (r=0.221, P=0.005), B (r=0.384, P<0.001), and S (r=0.467, P<0.001), MMP-2 (r=0.180, P<0.001), MMP-9 (r=0.178, P<0.001), and the number of lymphocytes (P<0.001) and plasma cells (P=0.001). OPG immunostaining was predominantly demonstrated in plasma cells. CONCLUSIONS: The concentration of aortic wall OPG is positively associated with established markers of AAA severity and pathogenesis. OPG appeared to be associated with lymphocytes and plasma cells. These human data support previous experimental data suggesting a role for OPG in AAA pathogenesis.

Proteasome inhibition with bortezomib depletes plasma cells and autoantibodies in experimental autoimmune myasthenia gravis.

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.

The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro and in vivo.

BACKGROUND: Multiple myeloma is characterized by the accumulation of tumor plasma cells in the bone marrow. Despite therapeutic improvements brought by proteasome inhibitors such as bortezomib, myeloma remains an incurable disease. In a variety of human cancers, human immunodeficiency virus protease inhibitors (e.g. nelfinavir) effectively inhibit tumor progression, but their impact on myeloma is unknown. We assessed the in vitro and in vivo effects of nelfinavir on multiple myeloma. DESIGN AND METHODS: The effects of nelfinavir (1-10 µM) on proteasome activity, proliferation and viability of myeloma cell lines and plasma cells from patients were assessed by measuring PERK, AKT, STAT3 and ERK1/2 phosphorylation and CHOP expression with immunoblotting or flow cytometry. The in vivo effect was assessed in NOD/SCID mice injected with luciferase expressing human myeloma cell lines and treated with nelfinavir at a dose of 75 mg/kg/day. Tumor progression was evaluated using a bioluminescent system. RESULTS: Nelfinavir inhibited 26S chymotrypsin-like proteasome activity, impaired proliferation and triggered apoptosis of the myeloma cell lines and fresh plasma cells. It activated the pro-apoptotic unfolded protein response pathway by inducing PERK phosphorylation and CHOP expression. Cell death triggered by nelfinavir treatment correlated with decreased phosphorylation of AKT, STAT3 and ERK1/2. Nelfinavir enhanced the anti-proliferative activity of bortezomib, dexamethasone and histone deacetylase inhibitors and delayed tumor growth in a myeloma mouse model. CONCLUSIONS: These results suggest that nelfinavir, used at a pharmacological dosage, alone or in combination, may be useful in the treatment of myeloma. Our data provide a preclinical basis for clinical trials using nelfinavir in patients with myeloma.

Inhibition of terminal differentiation of B cells mediated by CD27 and CD40 involves signaling through JNK.

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream events common to both receptors.

Proteasome inhibitor treatment of antibody-mediated allograft rejection.

PURPOSE OF REVIEW: Bortezomib is a first-in-class proteasome inhibitor that was originally Food and Drug Administration approved for the treatment of multiple myeloma. In the past few years, off-label use in solid organ transplant recipients has demonstrated its ability to provide plasma cell-targeted therapy in humans. The purpose of this review is to provide an update of recent basic science and clinical results with bortezomib in treating antibody-mediated rejection (AMR) that occurs in solid organ transplant recipients. RECENT FINDINGS: Proteasome inhibitor therapy for AMR in kidney transplant recipients is effective both as primary and as rescue therapy. Optimal responses with proteasome inhibitor therapy are obtained when AMR is diagnosed promptly and early in the posttransplant period. However, proteasome inhibitor therapy for late AMR (i.e., occurring 6 months or later posttransplant) provides less predictable results, likely due to the existence of a substantial bone marrow niche-resident long-lived plasma cell population. Proteasome inhibitor therapy has also recently been shown to provide effective therapy for AMR in heart, and also, transplant recipients. SUMMARY: Proteasome inhibitor therapy with bortezomib provides effective treatment for AMR in solid organ transplant recipients. As the first plasma cell-targeted therapy, proteasome inhibitor therapy provides the additional advantage of opening new possibilities for biologically defined plasma cell-targeted therapies.

The indoleamine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell-induced adaptive T regulatory cell generation.

Human plasmacytoid dendritic cells (PDCs) can drive naive, allogeneic CD4(+)CD25(-) T cells to differentiate into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). However, the intracellular mechanism or mechanisms underlying PDC-induced Treg generation are unknown. In this study, we show that human PDCs express high levels of IDO, an intracellular enzyme that catabolizes tryptophan degradation. Triggering of TLR 9 with CpG oligodeoxynucleotides activates PDCs to up-regulate surface expression of B7 ligands and HLA-DR Ag, but also significantly increases the expression of IDO and results in the generation of inducible Tregs from CD4(+)CD25(-) T cells with potent suppressor cell function. Blocking IDO activity with the pharmacologic inhibitor 1-methyl-D-tryptophan significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine, the immediate downstream metabolite of tryptophan, bypasses the 1-methyl-D-tryptophan effect and restores PDC-driven Treg generation. Our results demonstrate that the IDO pathway is essential for PDC-driven Treg generation from CD4(+)CD25(-) T cells and implicate the generation of kynurenine pathway metabolites as the critical mediator of this process.