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.

EPA and DHA have selective toxicity for PBMCs from multiple myeloma patients in a partly caspase-dependent manner.

Poly-unsaturated fatty acids (PUFAs) have been shown to have cytotoxic effects in both solid and non-solid tumors. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are among the most studied PUFAs. The aim of the present study was to evaluate the cytotoxic effects of these two fatty acids (FAs) in the peripheral blood mononuclear cells (PBMCs) obtained from untreated patients (new cases) with confirmed symptomatic multiple myeloma (MM). Our results showed that EPA at the concentration of 100 µM and DHA at 50 and 100 µM induce potent apoptotic effects in the PBMCs of MM patients (P < 0.05) as evidenced by Annexin V and propidium iodide (PI) staining, while they have little or no effects on the PBMCs isolated from healthy donors (P > 0.05). The observed effects were concentration- and time-dependent and 72 h treatment with DHA at a concentration of 100 µM had the strongest effect (P < 0.01). CD138 + cells isolated from MM patients showed great sensitivity to EPA/DHA. EPA- and DHA-induced apoptosis was significantly inhibited by the pan-caspase inhibitor (Z-VAD-FMK), indicating that cell death was at least partly dependent on caspase activation. The results of the present study showed that EPA and DHA have selective toxicities for malignant human plasma cells from MM patients, but not for mononuclear cells of healthy donors. These results warrant further studies with larger study populations to investigate the usefulness of PUFAs as a promising adjunctive therapy in the treatment of MM.

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.

RECQ1 helicase is involved in replication stress survival and drug resistance in multiple myeloma.

Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the expansion of multiple myeloma cells (MMCs) in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the most downregulated genes. RecQ helicases are DNA unwinding enzymes involved in the maintenance of chromosome stability. Here we show that RECQ1 is significantly overexpressed in MMCs compared to normal plasma cells and that increased RECQ1 expression is associated with poor prognosis in three independent cohorts of patients. Interestingly, RECQ1 knockdown inhibits cells growth and induces apoptosis in MMCs. Moreover, RECQ1 depletion promotes the development of DNA double-strand breaks, as evidenced by the formation of 53BP1 foci and the phosphorylation of ataxia-telangiectasia mutated (ATM) and histone variant H2A.X (H2AX). In contrast, RECQ1 overexpression protects MMCs from melphalan and bortezomib cytotoxicity. RECQ1 interacts with PARP1 in MMCs exposed to treatment and RECQ1 depletion sensitizes MMCs to poly(ADP-ribose) polymerase (PARP) inhibitor. DNMT inhibitor treatment results in RECQ1 downregulation through miR-203 deregulation in MMC. Altogether, these data suggest that association of DNA damaging agents and/or PARP inhibitors with DNMT inhibitors may represent a therapeutic approach in patients with high RECQ1 expression associated with a poor prognosis.

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.

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.

Tissue-infiltrating plasma cells are an important source of carboxylesterase 2 contributing to the therapeutic efficacy of prodrugs.

Carboxylesterase 2 (CES-2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. CES-2 expression was analyzed by immunohistochemistry in colorectal cancer (CRC) compared to colonic inflammation as well as in liver and peripheral blood. In CRC, tumor grades showed no correlation with levels of CES-2 expression, which was heterogeneous within these tumors. Cellular infiltrates in the immediate tumor vicinity expressed high levels of CES-2. Thus, tissue adjacent to the tumor was a substantial source of CES-2 with high expression in plasma cells. CES-2(high) plasma cells were abundantly found in the colon of patients with inflammatory bowel disease. CES-2 expression is strong in hepatocytes of normal livers, while CES-2 expression in peripheral blood mononuclear cells of healthy donors was overall low at protein and mRNA levels. In summary, the conversion of ester-containing prodrugs by CES-2 is mainly to occur in the periphery, during liver passage and in the colon after enterohepatic recirculation. We here demonstrated plasma cells as strong producers of CES-2. Further studies should elucidate the role of CES-2(+) plasma cells in intestinal inflammation and cancer.

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.

Insight into human protease activated receptor-1 as anticancer target by molecular modelling.

Protease-activated receptor 1 (PAR1) has been established as a promising target in many diseases, including various cancers. Strong evidence also suggests its role in metastasis. It is proved experimentally that PAR1 can induce numerous cell phenotypes, i.e. proliferation and differentiation. A strong link between PAR1 gene overexpression and high levels of ß-catenin was suggested by a study of the PAR1-Gα(13)-DVL axis in ß-catenin stabilization in cancers. An in vitro study was carried out to analyze PAR1 expression by flow cytometry on CD38+138+ plasma cells obtained from patients either at diagnosis (n: 46) (newly diagnosed multiple myeloma (NDMM)) or at relapse (n: 45) (relapsed/refractory multiple myeloma (RRMM)) and compared with the controls. Our previously synthesized benzoxazole (XT2B) and benzamide (XT5) derivatives were tested with in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, which revealed significant inhibitory activity on PAR1. We provide docking studies using Autodock Vina of these newly tested compounds to compare with the known PAR1 inhibitors in order to examine the binding mechanisms. In addition, the docking results are validated using HYDE binding assessment and a neural network (NN) scoring function.

Peroxidase-positive Auer bodies in plasma cells in multiple myeloma: a case report.

Reports of clinical cases with Auer bodies in the plasma cells in multiple myeloma (MM) are rare; however, most of those reported contain peroxidase (POX)-negative Auer bodies rather than the POX-positive Auer bodies observed in myeloid progenitors, indicating differences in their chemical properties. Furthermore, the cases with POX-positive Auer bodies similar to those observed in myeloid cells are extremely rare in non-myeloid cells. Here, we report the clinical features, laboratory investigations, diagnosis and treatment of a case of MM with POX-positive Auer bodies in plasma cells and review related the literature to advance the prognostic evaluation, diagnosis and treatment of similar cases.

Increased frequency of a unique spleen tyrosine kinase bright memory B cell population in systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is characterized by B cell hyperactivity and autoantibody production. As spleen tyrosine kinase (Syk) is pivotal in B cell activation, these experiments aimed to examine the extent to which Syk was abnormally expressed in SLE B cells and the nature of the B cell subset that differently expressed Syk. METHODS: B cells from healthy donors and SLE patients were analyzed by flow cytometry to assess basal expression of Syk and phosphorylated Syk. B cell subsets expressing higher levels of Syk were found, and their detailed phenotype, in vitro differentiation into plasmablasts/plasma cells, and Syk induction by cytokines were determined. RESULTS: Syk expression was higher in CD27+ memory B cells than in naive B cells from SLE patients. However, a significantly increased frequency of CD27- B cells with bright expression of Syk (Syk++) was found in SLE patients. CD27-Syk++ B cells showed enhanced basal expression of p-Syk and stronger Syk phosphorylation upon B cell receptor (BCR) engagement as compared to CD27-Syk+ B cells. CD27-Syk++ B cells were CD38- as well as CD19++, CD20++, and mainly CD21-, with decreased ABCB1 transporter activity. In contrast to CD27-Syk+ B cells, CD27-Syk++ B cells exhibited enhanced differentiation into CD27++ IgG-secreting cells and expressed somatically mutated BCR gene rearrangements. Syk++ B cells were inducible in vitro by stimulation with interferon-γ, lipopolysaccharide, or tumor necrosis factor α. CONCLUSION: SLE patients exhibit an increased frequency of hitherto unknown CD27-Syk++ memory-like B cells, indicating that intracellular Syk density could distinguish CD27- memory B cells from truly naive B cell subsets. Furthermore, the CD27-Syk++ subset is a candidate for a source of increased plasma cells in SLE.

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.

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.

[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.

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.

The natural pesticide dihydrorotenone induces human plasma cell apoptosis by triggering endoplasmic reticulum stress and activating p38 signaling pathway.

Dihydrorotenone (DHR) is a natural pesticide widely used in farming industry, such as organic produces. DHR is a potent mitochondrial inhibitor and probably induces Parkinsonian syndrome, however, it is not known whether DHR is toxic to other systems. In the present study, we evaluated the cytotoxicity of DHR on human plasma cells. As predicted, DHR impaired mitochondrial function by decreasing mitochondrial membrane potential in plasma cells. Because mito-dysfunction leads to unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, we examined the signature proteins in ER stress, including GRP78, ATF4, and CHOP. After DHR treatment, these proteins were significantly upregulated. It is reported that activation of the mitogen-activated protein kinases p38 and JNK are involved in endoplasmic reticulum stress. However, in the subsequent study, DHR was found to activate p38 but not the JNK signaling. When pre-treated with p38 inhibitor SB203580, activation of p38 and cell apoptosis induced by DHR was partially blocked. Thus, we found that DHR induced human plasma cell death by activating the p38 but not the JNK signaling pathway. Because plasma cells are very important in the immune system, this study provided a new insight in the safety evaluation of DHR application.

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.

The proteasome in terminal plasma cell differentiation.

The ability of eukaryotic cells to adapt to changing environmental conditions, respond to stimuli, and differentiate relies on their capacity to control the concentration, conformation, localization, and interaction of proteins, thereby reshaping their proteome. Protein degradation plays a critical role in maintaining protein homeostasis, and hence is carefully regulated. During the spectacular and demanding metamorphosis of activated B lymphocytes, expression programs are launched in coordinated waves, and adaptive strategies are deployed to prepare for antibody secretion. Surprisingly, though, despite increased demand for proteolysis, proteasome capacity collapses. As a result, antibody-secreting cells show symptoms of proteotoxic stress, and become extremely vulnerable to proteasome inhibition. The emerging concept that proteostenosis naturally follows B-cell activation has biological and immune implications, for it provides a model to dissect the integrated regulation of protein homeostasis, and a molecular counter limiting antibody responses, of use against autoimmune diseases. Mounting evidence linking proteotoxicity with proteasome vulnerability in malignant plasma cells visualizes strategies to understand responsiveness and obviate resistance to proteasome inhibition, with implications for the biology and therapy of plasma cell dyscrasias, namely, light chain amyloidosis and multiple myeloma.

Proteasome inhibition for antibody-mediated allograft rejection.

Antibody-mediated rejection (AMR) is a major risk factor for graft loss following kidney transplantation. Traditional anti-humoral therapies provide suboptimal therapy and they do not deplete plasma cells, which are the source of antibody production. Proteasome inhibitors (PI) have been shown to deplete both transformed and nontransformed plasma cells in human transplant recipients and animal models; and therefore, offer a new paradigm for AMR, ie, plasma cell-targeted therapy. Bortezomib, a first in class proteasome inhibitor approved by the US Food and Drug Administration for treatment of multiple myeloma, has been used to treat AMR in several solid organ transplant recipients. The greatest experience with PI therapy for treating AMR is in kidney transplant recipients. Experiences to date with PI therapy have demonstrated that: (1) early AMR (within the first 6 months post-transplant) responds better than late AMR, and (2) the nature of the plasma cell clonal population influences sensitivity to PI therapy with plasma subsets greater than long-lived bone marrow niche-resident plasma cells. In conclusion, plasma cell-targeted therapy with PIs is a method for targeting plasma cells (the source of antibody production) with a well-elucidated mechanism of action and subsequent points of synergy, thereby providing an exciting new potential means for enhancing anti-humoral therapies.

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.