scholarly journals Ultrastructural studies of human lymphoid cells. mu and J chain expression as a function of B cell differentiation.

1983 ◽  
Vol 158 (6) ◽  
pp. 1993-2006 ◽  
Author(s):  
I Hajdu ◽  
Z Moldoveanu ◽  
M D Cooper ◽  
J Mestecky
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Golgi Apparatus ◽  
B Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Peripheral Blood Lymphocytes ◽  
B Cell Differentiation ◽  
J Chain ◽  
Stimulation Of

J chain expression was examined as a function of the stage in differentiation along the B cell axis in humans. Intracellular distribution of J and mu chains in leukemic HLA-DR+ null and pre-B cells, and in normal B cells stimulated with pokeweed mitogen (PWM) was determined by immunoelectron microscopy and radioimmunoassay (RIA). J chain was detected in leukemic null and pre-B cells on free and membrane-bound ribosomes in the cytoplasm, or on perinuclear cisternae. Mu chain was found on free ribosomes and ribosomal clusters in leukemic pre-B cells but was absent in the leukemic null cells. In pre-B cell lines, mu chain was seen within rough endoplasmic reticulum (RER) and the Golgi apparatus whereas J chain was not detected in these organelles. However, both mu and J chain were detected in RER and the Golgi apparatus of immature and mature plasma cells induced by PWM stimulation of normal peripheral blood lymphocytes. Low levels of J chain were also detected by RIA in lysates of leukemic null and pre-B cells. Most of the intracellular J chain became detectable after reduction and alkylation of cell lysates, and free J chain was not found in the culture supernatants. The amount of intracellular and secreted immunoglobulin-bound J chain increased dramatically after PWM stimulation of peripheral blood lymphocytes. The majority of J chain-positive cells seen over an 8 d culture interval were lymphocytes and lymphoblasts, while mu chain was found primarily in plasma cells. These results suggest that J chain expression precedes mu chain synthesis during B cell differentiation and that a combination of the two chains for secretion is not initiated until the onset of plasma cells maturation.

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.

Gene Expression in Leukemic Blasts Persisting at Day 8 of Induction Therapy in Childhood ALL Displays a Common Shift towards Terminally Differentiated B Cells and a Cytoreduction-Associated Impairment of the Translational Machinery.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 537-537
Author(s):  
Peter Rhein ◽  
Stefanie Scheid ◽  
Richard Ratei ◽  
Christian Hagemeier ◽  
Karl Seeger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Induction Therapy ◽  
Peripheral Blood ◽  
Therapy Resistance ◽  
B Cell Differentiation ◽  
Translational Machinery ◽  
Blast Cells

Abstract In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. To approach the mechanisms of therapy resistance, we addressed genome-wide gene expression in blasts persisting after one week of induction therapy (day 8 blasts) and their molecular signatures as compared with blast cells at initial diagnosis (day 0 blasts). In order to approach this issue experimentally, a procedure has been established including flow sorting of leukemic blasts by their leukemia-associated immunophenotype and preparation of cRNA, starting from a small number of cells. Blast cells from 12 patients with precursor B-cell ALL were investigated using Affymetrix HG U133A microarrays, and genes commonly up- or down-regulated in blast cells under therapy were identified in matched pairs of day 8 and day 0 samples. In spite of the heterogeneous clinical features of the patients (mean rate of cytoreduction after 7 days of initial therapy = 82%, range between 33% and 99%), we were able to determine a set of 310 genes whose expression was commonly changed between day 8 and day 0 with an estimated false discovery rate of 0.05. The identified set of genes indicated inhibited cell cycling, reduced metabolism, and expression changes of multiple factors related to B-cell differentiation. These changes collectively suggested that gene expression in day 8 blasts is shifted towards resting mature B cells. To test this hypothesis, we isolated normal B cells from peripheral blood samples of leukemic patients and compared their gene expression to that of leukemic blasts using Principal Component Analysis (PCA). PCA revealed that day 8 samples are positioned between day 0 samples and normal B-cell samples, and statistical significance of this observation could be established using the Jonckheere-Terpstra test. Changes of B-cell differentiation markers on protein level supported this finding. In addition, we analyzed all genes with regard to the correlation of their expression changes with the rates of cytoreduction in peripheral blood. We observed differential impairment of the key components of the translational machinery including ribosome, eukaryotic 43S preinitiation complex and eukaryotic 48S initiation complex. Overall, expression levels of these factors decreased in therapy-sensitive patients but did not change in therapy-resistant patients. Taken together, investigation of leukemia cells persisting during therapy identifies common and individual expression changes which may potentially affect sensitivity towards anti-leukemic agents and offers new insights into the mechanisms of therapy resistance in ALL.

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.

BCR-mediated uptake of antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation

Blood ◽  
2009 ◽  
Vol 113 (17) ◽  
pp. 3969-3977 ◽  
Author(s):  
Julia Eckl-Dorna ◽  
Facundo D. Batista
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
Specific Class ◽  
B Cell Differentiation ◽  
Enhanced Production ◽  
Stimulation Of

Abstract The activation of Toll-like receptor 9 (TLR9) expressed within B cells is associated with enhanced humoral immunity. However the role of TLR9 in the stimulation of B-cell responses, and more specifically in shaping the outcome of B-cell differentiation, remains unclear. Here, we observed that immunization with the TLR9 agonist CpG linked to protein antigen gave rise to enhanced production of antigen-specific class-switched antibodies in vivo. Unlike dendritic cells, B cells are unable to acquire these conjugates by macropinocytosis and instead depend on uptake through a signaling-competent B-cell receptor (BCR), provided the overall BCR-antigen avidity exceeds a defined threshold. The resultant stimulation of intrinsic TLR9 leads to enhanced antigen-specific B-cell proliferation and differentiation to form extrafollicular plasma cells. Thus, the direct conjugation of antigen and CpG reveals a mechanism that may operate during the initiation of primary immune responses, and may prove useful as a strategy for the design of adjuvants suitable for vaccinations.

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.

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 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.

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