FRI0379 Metformin Suppresses Systemic Autoimmunity in Roquinsan/San Mice Through Inhibition of B Cell Differentiation into Plasma Cells VIA AMPK/MTOR/Stat3 Pathway Regulation

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.

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

Blood ◽

10.1182/blood.v83.8.2206.2206 ◽

1994 ◽

Vol 83 (8) ◽

pp. 2206-2210 ◽

Cited By ~ 6

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.20131831 ◽

2014 ◽

Vol 211 (5) ◽

pp. 827-840 ◽

Cited By ~ 52

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.

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

Blood Advances ◽

10.1182/bloodadvances.2019001279 ◽

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.

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Phenotypic and functional analysis of B cell lines from patients with multiple myeloma

Blood ◽

10.1182/blood.v66.2.444.bloodjournal662444 ◽

1985 ◽

Vol 66 (2) ◽

pp. 444-446

Author(s):

M Goldstein ◽

J Hoxie ◽

D Zembryki ◽

D Matthews ◽

AI Levinson

Keyword(s):

Multiple Myeloma ◽

Functional Analysis ◽

Cell Differentiation ◽

Cell Growth ◽

B Cell ◽

Cell Lines ◽

Plasma Cells ◽

Plaque Assay ◽

Functional Markers ◽

B Cell Differentiation

We characterized phenotypic and functional properties of B cell lines obtained from patients with multiple myeloma to determine how well they conform to particular stages of B cell differentiation. This information is a prerequisite for using such lines as tools for studying B cell growth and the regulation thereof. Two lines, GM1312 and GM1500, expressed B1 and Ia, determinants on early B cells, but expressed little, if any, T10, a determinant expressed on plasma cells. By contrast, B1 and Ia were poorly expressed on two other lines, GM2132 and U266. T10 was expressed on GM2132 but not on U266. Using a reverse hemolytic plaque assay, we also assessed the numbers of cells actively secreting immunoglobulin (IgSCs) in such cultures to provide a functional marker of B cell differentiation. We observed consistently higher numbers of IgSCs in cultures of GM2132 than in GM1500 and GM1312. These phenotypic and functional markers were stable over several months. The data suggest that such cell lines represent early (GM1312, GM1500) and later stages (GM2132, U266) of B cell differentiation, although all lines were derived from patients with multiple myeloma.

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Dietary Supplementation With Eicosapentaenoic Acid Inhibits Plasma Cell Differentiation and Attenuates Lupus Autoimmunity

Frontiers in Immunology ◽

10.3389/fimmu.2021.650856 ◽

2021 ◽

Vol 12 ◽

Author(s):

Azusa Kobayashi ◽

Ayaka Ito ◽

Ibuki Shirakawa ◽

Atsushi Tamura ◽

Susumu Tomono ◽

...

Keyword(s):

Fatty Acid Composition ◽

Fatty Acid ◽

Cell Differentiation ◽

Eicosapentaenoic Acid ◽

B Cell ◽

Acid Composition ◽

Plasma Cells ◽

Membrane Dynamics ◽

B Cell Differentiation ◽

Autoantibody Production

Accumulating evidence suggests that cholesterol accumulation in leukocytes is causally associated with the development of autoimmune diseases. However, the mechanism by which fatty acid composition influences autoimmune responses remains unclear. To determine whether the fatty acid composition of diet modulates leukocyte function and the development of systemic lupus erythematosus, we examined the effect of eicosapentaenoic acid (EPA) on the pathology of lupus in drug-induced and spontaneous mouse models. We found that dietary EPA supplementation ameliorated representative lupus manifestations, including autoantibody production and immunocomplex deposition in the kidneys. A combination of lipidomic and membrane dynamics analyses revealed that EPA remodels the lipid composition and fluidity of B cell membranes, thereby preventing B cell differentiation into autoantibody-producing plasma cells. These results highlight a previously unrecognized mechanism by which fatty acid composition affects B cell differentiation into autoantibody-producing plasma cells during autoimmunity, and imply that EPA supplementation may be beneficial for therapy of lupus.

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Plasma cell myeloma--new biological insights and advances in therapy

Blood ◽

10.1182/blood.v73.4.865.bloodjournal734865 ◽

1989 ◽

Vol 73 (4) ◽

pp. 865-879 ◽

Cited By ~ 1

Author(s):

B Barlogie ◽

J Epstein ◽

P Selvanayagam ◽

R Alexanian

Keyword(s):

Cell Differentiation ◽

B Cell ◽

Disease Progression ◽

Plasma Cell ◽

Plasma Cells ◽

Response To Therapy ◽

Myelogenous Leukemia ◽

Major Advance ◽

Plasma Cell Myeloma ◽

B Cell Differentiation

Plasma cell myeloma is a more complex neoplasm than suggested by the relative uniformity of its dominant plasma cells, which represent the terminal stage of normal B-cell differentiation. Phenotypic, molecular, and cellular genetic data favor the presence of a myeloma stem cell early in hematopoietic development so that, as in chronic myelogenous leukemia (CML), a far distance exists between the primordial malignant cell that was the target of malignant transformation and the dominant clinical phenotype. Traces of pre-B, myeloid, and T cells are coexpressed with the mature B-cell phenotype, an occurrence unknown in normal B-cell differentiation. Analogous to CML, disease progression is marked by disease dedifferentiation, occasionally with cessation of myeloma protein production and development instead of extramedullary lymphomalike features with high LDH or myelodysplasia/acute myelogenous leukemia (AML) syndromes. The prognostic importance of serum LDH levels even in newly diagnosed myeloma suggests the early presence of tumor cells with “LDH phenotype,” which, as a result of drug resistance and proliferative advantage, expand preferentially during disease progression. Further characterization of these cells may provide important clues about the ontogeny of multiple myeloma. Myeloma cells express many receptors for different biological signals that might be exploitable for therapy with immunotoxins or radioisotopes. Plasma cells and their precursors also produce a variety of cytokines, some of which have putatively autostimulatory functions (eg, IL-1, IL-5, IL-6) and/or are related to disease manifestations (eg, IL-1 and TNF-beta as OAF). The wealth of cellular expression by plasma cells provides clues for understanding the mechanisms of gene activation and the nature of abnormal growth and differentiation. The accuracy of prognostically relevant staging systems has been refined with the use of new quantitative parameters that reflect tumor mass (ie, serum B2M levels) and biology. Further studies of cellular and molecular biology (ie, CAL- LA, H-ras) may reveal those tumor cell features that define clinical entities, response to therapy, and long-term prognosis. The lack of a major advance in prognosis despite the use of more drugs and more intensive regimens justifies the continued use of standard melphalan- prednisone for patients with a highly favorable prognosis, for the very aged, and for those with a short life expectancy due to other major medical problems. However, a radical departure from standard practice is required to improve the prognosis for younger patients with poor risk features.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Blood ◽

10.1182/blood.v114.22.2946.2946 ◽

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.

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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 ◽

10.1182/blood.v82.9.2781.2781 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2781-2789 ◽

Cited By ~ 29

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.

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