Up-regulation Of Blimp-1 Research Articles

B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 Probably Through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation

Abstract 1109Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C). [Display omitted] Disclosures:Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

Recurrent 11q24.3 Gain Contributes to the Pathogenesis of Diffuse Large B Cell Lymphoma (DLBCL) by Deregulating ETS1 and FLI1

Abstract 1386DLBCL represents the most common form of B-cell non-Hodgkin lymphoma (B-NHL). It is an aggressive and heterogeneous disease, comprising at least three distinct subtypes based on gene expression profile analysis: germinal center B cell-like DLBCL (GCB), activated B cell-like DLBCL (ABC) and primary mediastinal B-cell lymphoma (PMBL). These subtypes are supposed to derive from B cells at different stages of differentiation. Normal germinal center (GC) B-cell differentiation requires a complex transcriptional program and alterations of genes involved in this process are relevant for DLBCL pathogenesis. Identification and functional characterization of new genetic lesions would provide critical information to better understand the pathogenesis of DLBCL. With this aim, we studied the genomic profiles of 166 DLBCL patients, identified and characterized a recurrent gain mapping to chromosome 11q24.3. Methods.Genomic profiles were obtained from 166 Affymetrix 250K SNP arrays and integrated with gene expression data (GeneChip U133 plus 2.0) in 54 cases. Data were validated by PCR and immunohistochemistry. Gene silencing experiments were done with shRNA. Results.A minimal common region 11q24.3 gain was present in 26% of DLBCL samples and it encompassed six genes (ETS1, FLI1, KCNJ1, KCNJ5, P53AIP1, RICS). Samples with the 11q24.3 gain were significantly associated with high expression of the transcription factors ETS1 and FLI1. Data were confirmed by real-time PCR and by immunohistochemical analysis. Gene expression analysis showed 228 transcripts with a significantly different expression between cases with or without the lesion (p<0.01, >2-fold change): 215 genes were up-regulated in the patients with the gain and 13 were down-regulated, suggesting that this lesion has an impact on the transcriptional program of the tumor cells. To study the biological meaning of the lesion, ETS1 and FLI1 expression was down-regulated in a DLBCL cell line bearing the same lesion observed in clinical specimens (OCI-Ly7). Results showed that ETS1 and FLI1 down-regulation caused a reduced proliferation rate and activation of apoptosis leading to cell death. Concomitant ETS1 and FLI1 down-regulation resulted in a more severe phenotype. Only FLI1 was confirmed to be essential for cell viability in other DLBCL cell lines (SUDHL4, VAL, U2932), whereas ETS1 did not, suggesting a distinct role of the two transcription factors in different DLBCL samples. Preliminary results showed that down-regulation of ETS1 affected the transcriptional program of GC B-cell terminal differentiation causing an up-regulation of BLIMP1, the master regulator of plasma cells differentiation. Conclusions.In DLBCL, a recurrent gain at 11q24.3 determines the over-expression of the transcription factors ETS1 and FLI1. Functional experiments showed that the lesion might sustain DLBCL proliferation and viability, and contribute to a differentiation blockade of the GC B-cell towards a plasma cell lineage by negatively regulating BLIMP1. Disclosures:No relevant conflicts of interest to declare.

PAX5 Phosphorylation by MAP Kinase Signal Potentially Suppresses PAX5 Function and Might Be a Trigger of the Plasma Cell Differentiation

AbstractAbstract 3898Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of Blimp1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of Blimp1 and XBP-1 and subsequent suppression of Pax5 by Blimp1 were observed and thought to be the trigger of plasma cell differentiation. However, the first trigger that represses PAX5 function is yet to be revealed. Here, we investigated the relevance between Pax5 and MAP kinase signal, a main component of BCR signal. We demonstrated PAX5 phosphorylation by ERK2 in vitro and in vivo (Figure. A). The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In the luciferase reporter assays, PAX5 phosphorylation by MAP kinase signal reduced the transcriptional activity of PAX5 when CD19 promoter sequence was used for reporter gene. On the other hand, PAX5 caused transcriptional repression of Blimp1 promoter, which was released by PAX5 phosphorylation by MAP kinase signal. These data supported the reported findings that PAX5 had dual function, and suggested that the PAX5 function was inhibited by its phosphorylation. We demonstrated that B cell receptor stimulation with anti-IgM antibody induced strong MAP kinase activation, phosphorylation of endogenous Pax5 (Figure. B), and upregulation of Blimp1 mRNA (Figure. C). All of these phenomena were inhibited by U0126, MEK inhibitor. These results suggested that transcriptional repression of Blimp1 by PAX5 was released by its phosphorylation by MAP kinase signal. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells. [Display omitted] Disclosures:Hayakawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.

Virus-like particle vaccine activates conventional B2 cells and promotes B cell differentiation to IgG2a producing plasma cells

We have previously shown that immunization with SIV-, SHIV-, or HA (influenza hemagglutinin)-virus-like particles (VLPs) elicits a strong humoral immune response in mice. However, little is known about the action VLPs exert on immune effector cells, including B cells. In this study, we found that all three types of VLPs could directly bind and activate B cells in vitro. VLPs stimulated the proliferation of B220+IgM+CD43−CD5− B2 cells and their differentiation to plasma cells that preferentially produce IgG2a antibodies. Up-regulation of Blimp-1, XBP-1, IRF4, and AID genes, which are responsible for class-switch recombination and somatic hypermutation, was observed in VLP-activated B2 cells. Stimulation of naïve splenocytes with VLPs led to a high expression of IL-12, RANTES and MIP, the cytokine milieu that favors B cell differentiation into IgG2a secreting cells. VLP immunization of C57BL/6 mice corroborated our in vitro data showing enlarged germinal centers and expanded conventional B2 cells, but no enlarged marginal zone B1 cells, in the spleen. Enhanced antigen-specific plasma cell formation, antibody production, and IgG2a class switching were found in VLP-immunized groups. The current study details the interaction between VLPs and B cells which result in preferential IgG2a antibody production following VLP immunization.

STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.

Different Kinetics of Blimp-1 Induction in B Cell Subsets Revealed by Reporter Gene

The transcriptional repressor Blimp-1 (B lymphocyte-induced maturation protein 1) has been described as a "master regulator" of B cell differentiation into Ab-secreting cells (ASCs). Although there is mounting evidence for the importance and necessity of Blimp-1 in plasma cell development, there is uncertainty as to the role it plays in B cell differentiation of B cell subsets and the way in which it may interact with other transcription factors such as Pax5 and Bcl6 during ASC differentiation. Using a mouse expressing GFP under the control of the Blimp-1 regulatory elements (Blimp-1(GFP/+)), we examined the kinetics of Blimp-1 up-regulation in purified B cell subsets following activation. B1 cells showed the most rapid and pronounced up-regulation of Blimp-1 in response to the mitogens tested, followed by marginal zone B cells and then conventional B2 cells. Interestingly, only B1 cells substantially up-regulated Blimp-1 expression in response to CpG. B1 cells secreted negligible Ig upon isolation but were able to up-regulate Blimp-1 and initiate Ig secretion within 28 h of stimulation. Also of interest, B1 cells have a transcriptional factor profile that is intermediate between a naive B cell and an ASC, indicative of the semiactivated state of B1 cells. Transferred naive Blimp-1(GFP/+) B1 and B2 cells both gave rise to ASCs in the bone marrow, suggesting no intrinsic barriers to B1 cell entry into the long-lived ASC compartment.

Survival and Proliferation Factors of Normal and Malignant Plasma Cells

Since the first identification of interleukin (IL)-6 as a myeloma cell growth factor by Dr. Kawano's and Dr. Klein's groups 14 years ago, numerous studies have emphasized its major roles in the emergence of malignant plasma cells in vivo and in the generation of normal plasma cells. Four transcription factors control B-cell differentiation into plasma cells. The B-cell transcription factor pax-5 is mainly responsible for a B-cell phenotype, and bcl-6 represses the plasma cell transcription factor blimp-1 and plasma cell differentiation. bcl-6 expression is triggered by CD40 and IL-4 activation. A lack of CD40 and IL-4 activation yields a down-regulation of bcl-6 expression, and IL-6 stimulation yields an up-regulation of blimp-1, mainly through STAT3 activation. Blimp-1 further down-regulates bcl-6 and pax-5 expression and makes plasma cell differentiation possible. IL-6 as well as IL-10 up-regulate XBP-1. XBP-1 is another transcription factor that is involved in plasma cell differentiation and whose gene expression is shut down by pax-5. The plasma cell transcription factors blimp-1 and XBP-1 are up-regulated, and the B-cell transcription factors bcl-6 and pax-5 are down-regulated, in malignant cells compared to B-cells. Apart from the recent identification of these 4 transcription factors, the factors involved in normal plasma cell generation are mostly unknown. Regarding malignant plasma cells, 3 categories of growth factors have been identified: (1) the IL-6 family cytokines, IL-10, and interferon alpha that activate the Janus kinase-signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase pathways; (2) growth factors activating the phosphatidylinositol (PI)-3 kinase/AKT and MAP kinase pathways, unlike the JAK/STAT pathway (insulin-like growth factor 1, hepatocyte growth factor, and members of the epidermal growth factor family able to bind syndecan-1 proteoglycan); and (3) B-cell-activating factor (BAFF) or proliferation-inducing ligand (APRIL) that activate the nuclear factor KB and PI-3 kinase/AKT pathways. BAFF and APRIL bind to BAFF receptor and TACI and are major B-cell survival factors. Recent data indicate that these various growth factors may cooperate to provide optimum signaling because they are localized together and with cytoplasmic transduction elements in caveolinlinked membrane caveolae. The identification of these myeloma cell growth factors and of the associated transduction pathways should provide novel therapeutic targets in multiple myeloma.

Tracking the response of Xid B cells in vivo: TI-2 antigen induces migration and proliferation but Btk is essential for terminal differentiation.

X-linked immunodeficient (Xid) mice carry a Bruton's tyrosine kinase (Btk) mutation and exhibit a selective failure to produce antibodies against bacterial capsular polysaccharides. Studies in vitro point to a fundamental survival defect of Xid B cells after receptor cross-linking by thymus-independent type-2 (TI-2) antigen because B cells undergo apoptosis without proliferating. We describe results from a novel model, which we have used to investigate the impact of the Xid mutation on migration, proliferation and differentiation of B cells after polysaccharide immunization in vivo. Immunoglobulin knock-in mice, in which a large proportion of B cells express transgene-encoded receptors specific for (4-hydroxy-3-nitrophenyl)-acetyl (NP), were crossed with CBA/N mice. The male progeny contain NP-specific Xid B cells, while the female progeny contain NP-specific B cells with normal Btk. After immunization with the TI-2 antigen NP-Ficoll, NP-specific Xid B cells migrate to the T zones and proliferate. Despite transient up-regulation of blimp-1 and survival beyond the time when terminal differentiation is normally underway, Btk-defective B cells fail to differentiate to plasmablasts or germinal center cells. CD40 ligation partially restores their ability to form plasma cells in response to TI-2 antigen.

Blimp-1 overcomes the block in IgM secretion in lipopolysaccharide/anti-mu F(ab')2-co-stimulated B lymphocytes.

A combination of signals transmitted through the antigen receptor, membrane-bound cell interaction molecules and cytokine receptors induces B cell proliferation and differentiation into immunoglobulin-secreting or memory cells. It has recently been suggested by Turner et al. (Cell 1994. 77: 297) that the complex changes in gene activities accompanying high levels of immunoglobulin secretion are under the common control of a master regulator, Blimp-1 (B lymphocyte-induced maturation protein). We show here that in naive mouse B cells stimulated with lipopolysaccharide (LPS) alone (which leads to high IgM production), Blimp-1 is highly expressed, while cells co-stimulated with LPS and anti-mu F(ab')2 show low levels of Blimp-1 mRNA and no longer secrete Ig. I gamma 1 sterile transcripts are, however, up-regulated after receptor co-ligation. Addition of interleukin (IL)-2 and IL-5 to LPS + anti-mu F(ab')2-treated primary B cells led to up-regulation of Blimp-1 and IgM secretion. Transfection of a Blimp-1 expression vector also induced IgM secretion. The data indicate that Blimp-1 is an important regulator of immunoglobulin secretion by primary B cells, and suggest that its level of expression may determine the differentiation to Ig-secreting plasma cells or entrance and maintenance in the memory pool.