Involvement of Ets Factor Spi-B, E Protein E2-2, and Id2 in Waldenström's Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2946-2946
Author(s):  
Yangsheng Zhou ◽  
Xia Liu ◽  
Lian Xu ◽  
Zachary Hunter ◽  
Jenny Sun ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
B Cell Differentiation ◽  
Protein Activity ◽  
E Proteins ◽  
E Protein ◽  
Nuclear Extracts

Abstract Abstract 2946 Poster Board II-922 Waldenström's macroglobulinemia (WM) is an incurable B cell disorder with a lymphoplasmacytic infiltrate in the bone marrow (BM) and IgM monoclonal gammopathy. WM tumor cells show variable differentiation, ranging from mature B-cells to plasma cells, which likely results from failure to fully undergo differentiation. In this study, we analyzed the expression of several genes involved in B cell differentiation by real time RT-PCR, such as Ets factors, the basic helix-loop-helix (bHLH) E proteins, as well as the inhibitors of DNA binding (Id) proteins which antagonize E protein activity. Comparison of BM CD19+ B cells obtained from 13 WM patients with 6 age-matched healthy donors showed that expression of the Ets factor Spi-B was increased four-fold, while Id2 was decreased three-fold. However, transcript levels of E proteins were similar between the two groups. Transduction of Spi-B in BCWM.1 WM cells resulted in two-fold higher levels of Id2 and five-fold lower levels of E2-2 compared with control. Id2 transduced BCWM.1 cells expressed two-fold lower levels of E2-2 and Spi-B. Taken together, these results implicate that increased expression of Spi-B alone cannot suppress Id2 transcription in the absence of E2-2 activity. Interestingly, overexpressing Spi-B while concomitantly knocking down Id2 increased the expression of the XBP-1 splicing isoform 2.5-fold without changing levels of Blimp-1 and IRF4. Moreover, inhibition of Spi-B expression by RNA interference or forced expression of Id2 in transduced BCWM.1 cells induced a significant decrease of anti-apoptotic Bcl-2. Importantly, we also showed that Spi-B co-immunoprecipated with Blimp-1 in nuclear extracts. Collectively, these data suggest that the regulatory network of the Spi-B, E2-2, and Id2 plays an essential role in B cell differentiation as well as the pathogenesis of WM, and suggests that Spi-B overexpression may block WM cell differentiation by sequestration of Blimp-1 while promoting tumor cell survival though up-regulation of Bcl-2. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dynamic changes in Id3 and E-protein activity orchestrate germinal center and plasma cell development

2016 ◽  
Vol 213 (6) ◽  
pp. 1095-1111 ◽  
Author(s):  
Renee Gloury ◽  
Dimitra Zotos ◽  
Malou Zuidscherwoude ◽  
Frederick Masson ◽  
Yang Liao ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Protein Activity ◽  
Dynamic Changes ◽  
Plasma Cell Differentiation ◽  
E Protein

The generation of high-affinity antibodies requires germinal center (GC) development and differentiation of long-lived plasma cells in a multilayered process that is tightly controlled by the activity of multiple transcription factors. Here, we reveal a new layer of complexity by demonstrating that dynamic changes in Id3 and E-protein activity govern both GC and plasma cell differentiation. We show that down-regulation of Id3 in B cells is essential for releasing E2A and E2-2, which in a redundant manner are required for antigen-induced B cell differentiation. We demonstrate that this pathway controls the expression of multiple key factors, including Blimp1, Xbp1, and CXCR4, and is therefore critical for establishing the transcriptional network that controls GC B cell and plasma cell differentiation.

Involvement of Ets Factor Spi-B and Id2 In Waldenström's Macroglobulinemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 129-129
Author(s):  
Yangsheng Zhou ◽  
Xia Liu ◽  
Lian Xu ◽  
Zachary Hunter ◽  
Jenny Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
B Cells ◽  
Plasma Cell ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
B Cell Differentiation ◽  
Protein Activity ◽  
E Proteins ◽  
Plasma Cell Differentiation

Abstract Abstract 129 Waldenström's macroglobulinemia (WM) is an incurable disorder with a lymphoplasmacytic infiltrate in the bone marrow (BM) and IgM monoclonal gammopathy. WM tumor cells show variable differentiation, ranging from mature B-cells to plasma cells, which likely results from failure to fully undergo differentiation. Here we analyzed the expression of several genes involved in B cell differentiation by real time RT-PCR, such as Ets factors, the basic helix-loop-helix (bHLH) E proteins, as well as the inhibitors of DNA binding (Id) proteins which antagonize E protein activity. Comparison of bone marrow CD19+ B cells obtained from 12 untreated WM patients with 15 age-matched healthy donors showed that expression of the Ets factor Spi-B was increased four-fold, while Id2 was decreased three-fold. However, transcript levels of E proteins were similar between these two groups. Furthermore, along with differentiation of primary human CD19+ cells from peripheral blood into CD38+CD20− plasmablasts, Spi-B and Id2 expression levels were significantly decreased and increased, respectively. Ectopic expression of Spi-B in primary human CD19+ cells inhibited the plasma cell differentiation associated with decreased transcription levels of BLIMP1, XBP-1 spliced form, and IRF4. In addition, overexpression of Spi-B in BCWM.1 WM cells also resulted in repressed expression of BLIMP1, XBP-1 spliced form, and IRF4. Conversely, knocking down Spi-B in BCWM.1 WM cells increased IRF4 and Id2 expression. Importantly, in primary WM bone marrow CD19+ cells, knocking down of Spi-B induced CD38+CD20- plasma cell formation and increased expression of BLIMP1, XBP-1 spliced, IRF4, and Id2. Moreover, knocking down Spi-B in primary WM cells decreased Bcl-2 expression. Collectively, our results suggest that Spi-B overexpression plays an essential role in the pathogenesis of WM by repressing factors involved in plasma cell differentiation while promoting tumor cell survival through Bcl-2. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

2021 ◽  
Author(s):  
Casper Marsman ◽  
Dorit Verhoeven
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
B Cell Differentiation ◽  
Differentiation Potential ◽  
Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

scholarly journals Modulation of spontaneous B-cell differentiation in macroglobulinemia by retinoic acid

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2206-2210 ◽  
Author(s):  
Y Levy ◽  
S Labaume ◽  
MC Gendron ◽  
JC Brouet
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Culture Supernatants

Abstract We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells. This process is dependent on an interleukin (IL)-6 autocrine pathway. We investigate here whether all-trans-retinoic acid (RA) interferes with B-cell differentiation either in patients with IgM gammapathy of undetermined significance (MGUS) or Waldenstrom's macroglobulinemia (WM). RA at a concentration of 10(-5) to 10(-8) mol/L inhibited by 50% to 80% the in vitro differentiation of purified B cells from four of five patients with MGUS and from one of five patients with WM as assessed by the IgM content of day 7 culture supernatants. We next determined whether this effect could be related to an inhibition of IL- 6 secretion by cultured B cells and/or a downregulation of the IL-6 receptor (IL-6R), which was constitutively expressed on patients' blood B cells. A 50% to 100% (mean, 80%) inhibition of IL-6 production was found in seven of 10 patients (five with MGUS and two with WM). The IL- 6R was no more detectable on cells from patients with MGUS after 2 days of treatment with RA and slightly downregulated in patients with WM. It was of interest that B cells susceptible to the action of RA belonged mostly to patients with IgM MGUS, which reinforces our previous data showing distinct requirements for IL-6-dependent differentiation of blood B cells from patients with VM or IgM MGUS.

scholarly journals The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity

2014 ◽  
Vol 211 (5) ◽  
pp. 827-840 ◽  
Author(s):  
Stéphane Chevrier ◽  
Dianne Emslie ◽  
Wei Shi ◽  
Tobias Kratina ◽  
Cameron Wellard ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Cell Cycle Progression ◽  
Plasma Cells ◽  
Cell Lineage ◽  
B Cell Differentiation ◽  
Antibody Secreting Cell ◽  
Important Player

The transcriptional network regulating antibody-secreting cell (ASC) differentiation has been extensively studied, but our current understanding is limited. The mechanisms of action of known “master” regulators are still unclear, while the participation of new factors is being revealed. Here, we identify Zbtb20, a Bcl6 homologue, as a novel regulator of late B cell development. Within the B cell lineage, Zbtb20 is specifically expressed in B1 and germinal center B cells and peaks in long-lived bone marrow (BM) ASCs. Unlike Bcl6, an inhibitor of ASC differentiation, ectopic Zbtb20 expression in primary B cells facilitates terminal B cell differentiation to ASCs. In plasma cell lines, Zbtb20 induces cell survival and blocks cell cycle progression. Immunized Zbtb20-deficient mice exhibit curtailed humoral responses and accelerated loss of antigen-specific plasma cells, specifically from the BM pool. Strikingly, Zbtb20 induction does not require Blimp1 but depends directly on Irf4, acting at a newly identified Zbtb20 promoter in ASCs. These results identify Zbtb20 as an important player in late B cell differentiation and provide new insights into this complex process.

scholarly journals TLR-mediated activation of Waldenström macroglobulinemia B cells reveals an uncoupling from plasma cell differentiation

2020 ◽  
Vol 4 (12) ◽  
pp. 2821-2836
Author(s):  
Jennifer Shrimpton ◽  
Matthew A. Care ◽  
Jonathan Carmichael ◽  
Kieran Walker ◽  
Paul Evans ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Immunoglobulin M ◽  
B Cell Differentiation ◽  
Waldenström Macroglobulinemia ◽  
Plasma Cell Differentiation ◽  
Waldenstrom Macroglobulinemia

Abstract Waldenström macroglobulinemia (WM) is a rare malignancy in which clonal B cells infiltrate the bone marrow and give rise to a smaller compartment of neoplastic plasma cells that secrete monoclonal immunoglobulin M paraprotein. Recent studies into underlying mutations in WM have enabled a much greater insight into the pathogenesis of this lymphoma. However, there is considerably less characterization of the way in which WM B cells differentiate and how they respond to immune stimuli. In this study, we assess WM B-cell differentiation using an established in vitro model system. Using T-cell–dependent conditions, we obtained CD138+ plasma cells from WM samples with a frequency similar to experiments performed with B cells from normal donors. Unexpectedly, a proportion of the WM B cells failed to upregulate CD38, a surface marker that is normally associated with plasmablast transition and maintained as the cells proceed with differentiation. In normal B cells, concomitant Toll-like receptor 7 (TLR7) activation and B-cell receptor cross-linking drives proliferation, followed by differentiation at similar efficiency to CD40-mediated stimulation. In contrast, we found that, upon stimulation with TLR7 agonist R848, WM B cells failed to execute the appropriate changes in transcriptional regulators, identifying an uncoupling of TLR signaling from the plasma cell differentiation program. Provision of CD40L was sufficient to overcome this defect. Thus, the limited clonotypic WM plasma cell differentiation observed in vivo may result from a strict requirement for integrated activation.

scholarly journals Human pre-B cells differentiate into Ig-secreting plasma cells in the presence of interleukin-4 and activated CD4+ T cells or their membranes

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2781-2789 ◽  
Author(s):  
J Punnonen ◽  
G Aversa ◽  
JE de Vries
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Cultures ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Interleukin 4 ◽  
B Cell Differentiation ◽  
Ig Synthesis

Abstract Studies on human B-cell development have been hampered by the lack of reproducible culture techniques to induce pre-B cells to differentiate into Ig-secreting plasma cells. Here, we describe that highly purified surface (s) mu-, cytoplasmic (c) mu+, CD10+, CD19+ human pre-B cells derived from fetal bone marrow (BM) differentiate with high frequencies into Ig-secreting plasma cells, when cocultured with activated, cloned CD4+ T cells and with interleukin-4 (IL-4). Production of IgM, total IgG, IgG4, and IgE in pre-B-cell cultures was detected, indicating that the cells also underwent Ig isotype switching. Pre-B-cell differentiation occurred in the absence of BM stromal cells, IL-7, and stem cell factor (SCF). However, IL-7 significantly enhanced the levels of Ig produced, whereas SCF was ineffective. Neutralizing anti-IL-4 monoclonal antibodies (MoAbs) completely inhibited pre-B-cell differentiation showing the specificity of the reaction. Intact CD4+ T- cell clones could be replaced by membrane preparations of these cells, indicating that the costimulatory signals provided by the activated CD4+ T cells are contact-mediated. In contrast, anti-CD40 MoAbs failed to provide the costimulatory signal required for pre-B-cell differentiation, which may be related to the very low expression of CD40 on fetal BM B cells. Activated CD4+ T cells and IL-4 also induced s mu expression and Ig synthesis in cultures initiated with pre-B cells that had been preincubated in medium for 2 days, and from which spontaneously emerging s mu+ B cells were removed by using a fluorescence-activated cell sorter. These results support the notion that the Ig synthesis observed in pre-B-cell cultures was not caused by outgrowth and differentiation of cells that spontaneously matured into s mu+ B cells. In addition, IL-4 and CD4+ T cells strongly enhanced CD40 and HLA-DR expression on the majority of cultured pre-B cells, further indicating that CD4+ T cells and IL-4 activate bona fide pre-B cells. Taken together, these data indicate that activated CD4+ T cells and IL-4 can provide all the necessary signals required for human pre-B cells to differentiate into Ig-secreting plasma cells.

scholarly journals Memory B Cells Are Biased Towards Terminal Differentiation: A Strategy That May Prevent Repertoire Freezing

1997 ◽  
Vol 186 (6) ◽  
pp. 931-940 ◽  
Author(s):  
Christophe Arpin ◽  
Jacques Banchereau ◽  
Yong-Jun Liu
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Clone ◽  
Terminal Differentiation ◽  
Memory B Cells ◽  
B Cell Differentiation

Isolation of large numbers of surface IgD+CD38− naive and surface IgD−CD38− memory B cells allowed us to study the intrinsic differences between these two populations. Upon in vitro culture with IL-2 and IL-10, human CD40–activated memory B cells undergo terminal differentiation into plasma cells more readily than do naive B cells, as they give rise to five- to eightfold more plasma cells and three- to fourfold more secreted immunoglobulins. By contrast, naive B cells give rise to a larger number of nondifferentiated B blasts. Saturating concentrations of CD40 ligand, which fully inhibit naive B cell differentiation, only partially affect that of memory B cells. The propensity of memory B cells to undergo terminal plasma cell differentiation may explain the extensive extra follicular plasma cell reaction and the limited germinal center reaction observed in vivo after secondary immunizations, which contrast with primary responses in carrier-primed animals. This unique feature of memory B cells may confer two important capacities to the immune system: (a) the rapid generation of a large number of effector cells to efficiently eliminate the pathogens; and (b) the prevention of the overexpansion and chronic accumulation of one particular memory B cell clone that would freeze the available peripheral repertoire.

scholarly journals Humoral helper activity for B-cell differentiation released from non- Hodgkin's lymphoma cells having both SRBC and complement receptors in the pokeweed mitogen system

Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1074-1080
Author(s):  
N Moriya ◽  
T Miyawaki ◽  
Y Ueno ◽  
S Koizumi ◽  
N Taniguchi
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Lymphoid Cells ◽  
Pokeweed Mitogen ◽  
Complement Receptors ◽  
B Cell Differentiation ◽  
Cell Leukemia

Abstract The majority of lymphoid cells from a patient with non-Hodgkin's lymphoma with leukemic transformation were demonstrated to carry receptors for both sheep erythrocytes and complements by the combined rosette assay using neuraminidase-treated sheep erythrocytes and complement-coated zymosan beads. Most of them were considered morphologically lymphoblasts and were positive for acid phosphatase staining. Terminal deoxynucleotidyl transferase activity was not detected in these cells. Lymphoid cells from this patient did not respond to the stimulation with phytohemagglutinin-P, concanavalin-A, and pokeweed mitogen (PWM). When these cells were cultured with PWM for 7 days, no plasma cell was generated. Although only a few plasma cells were generated in the PWM-stimulated culture of normal purified B cells alone, the addition of the patient's cells to purified normal B cells resulted in a markedly enhanced generation of plasma cells in response to PWM, as was the case with normal T cells. But leukemic cells either from a patient with T-cell leukemia not having complement receptors or from a patient with null-cell leukemia showed no enhancing ability in B- cell differentiation. In addition, the culture supernates of the patient's cells obtained after 24-hr PWM stimulation had an ability to promote B-cell differentiation comparable in activity to those from the PWM-stimulated normal T cells.

scholarly journals A novel enhancer, the pro-B enhancer, regulates Id1 gene expression in progenitor B cells.

1995 ◽  
Vol 15 (3) ◽  
pp. 1513-1521 ◽  
Author(s):  
S Saisanit ◽  
X H Sun
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Regulatory Elements ◽  
B Cell Differentiation ◽  
Protein Activity ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Id Proteins

The helix-loop-helix (HLH) Id proteins have been reported to function as inhibitors of various differentiation programs. The HLH motif mediates dimer formation between Id and the basic HLH transcription factors. Since Id proteins lack the basic region responsible for DNA binding, the heterodimers cannot bind to DNA. Id proteins have also been found to be involved in early B-cell differentiation. They are expressed at high levels in progenitor B cells (pro-B cells), and the expression is diminished in pre-B cells and mature B cells. This expression pattern correlates inversely with basic HLH protein activity and immunoglobulin enhancer function in B-cell development. Regulation of Id expression may play an important role in transcriptional control of immunoglobulin genes and therefore in B-cell differentiation. We have characterized the regulatory elements of the Id1 gene. Using stable transfectants, transient transfection, and mobility shift assays, we have identified an 8-bp element designated PBE (pro-B enhancer) downstream of the Id1 gene that is responsible for a pro-B-cell-specific enhancer activity. A pro-B-cell-specific protein complex was found to bind to the 8-bp PBE element. Substitution mutagenesis at this binding site showed that it is indeed of functional importance in regulating the pro-B-cell-specific expression of the Id1 gene.

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