Over-Expression of IRF4 (MUM1) Protein Predicts Poorer Outcome in Myeloma Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5127-5127
Author(s):  
Ruth M de Tute ◽  
Reuben Tooze ◽  
Roger G Owen ◽  
Andy C Rawstron
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Populations ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Over Expression ◽  
Cell Purification

Abstract IRF4 (also known as MUM1) is a member of the interferon regulatory factor family of transcription factors whose expression is critical for the transition from pre B-cell to immature B-cell and for differentiation of mature B-cell to plasma cell. It has previously been reported by Heintel el al (Leukemia (2008) 22, 441–445) that over-expression of IRF4 at the mRNA level is associated with a poor outcome in myeloma. Measurement of expression at the protein level using flow cytometry would remove the need for plasma cell purification and also allow evaluation of IRF4 in other cell populations. We have developed a multi-colour flow cytometry assay incorporating an indirect intracellular staining using the goat polyclonal antibody IRF4 (Santa Cruz). This permits simultaneous analysis of IRF4 in combination with five cell surface markers, allowing quantitation of the IRF4 levels present in specific B-cell and plasma cell populations. The aim of this study was to assess the expression profile of IRF4 in neoplastic and normal B-cells and plasma cells and evaluate the relationship between IRF4 protein expression and outcome in multiple myeloma patients. Leucocytes prepared from whole bone marrow using ammonium chloride lysis were incubated with surface markers CD52 PE, CD38 PerCPCy5.5, CD19 Pe-Cy7, CD138 APC and CD20 APC-Cy7. Cells were then fixed and permeabilised before incubation with unconjugated IRF4, washing and incubation with an Alexa488 secondary antibody. IRF4 expression showed the expected profile in samples with normal B-lineage cells. Low levels of expression were found in normal mature B-cells (mean MFI 1033, range 395–2265), higher levels in progenitor B-cells (mean 4.1-fold higher than mature B-cells, mean MFI 4252, range 612–22958) and strong uniform expression in normal BM plasma cells (mean 13.8-fold higher than mature B-cells, mean MFI 14250, range 7391–21914). Plasma cells from untreated myeloma patients showed a mean 1.9-fold higher IRF4 expression level than normal plasma cells (P=<0.001, normal n=16, neoplastic n = 19, mean MFI 27510, range 6807–53662). The myeloma patients were then separated into two groups; those with IRF4 expression in the normal range (n=6, mean MFI 12858, range 4182–21028) and those with high IRF4 expression (n=13, mean MFI 34501, range 24892–53662). After a median follow-up of 19 months, 6 patients had died in the high expression group (46.2%) compared with no deaths in the low expression group; this difference was statistically significant (Cox regression analysis, p=0.036). The assay developed can detect quantitative differences in IRF4 expression between B-progenitors, mature B-cells, normal plasma cells and neoplastic plasma cells. The ability to simultaneously analyse IRF4 expression with other cell surface markers allows rapid enumeration of IRF4 protein levels without plasma cell purification and greatly enhances the potential for IRF4 to be used as a prognostic marker. Neoplastic plasma cells can be categorised as having low or high levels of IRF4 expression with over-expression predicting a significantly shorter overall survival.

Humoral Immunity and Plasma Cell Changes in Patients Responding to CD19-Specific Chimeric Antigen Receptor (CAR)-Modified T-Cell Adoptive Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1110-1110
Author(s):  
Vijay Bhoj ◽  
Michael C Milone ◽  
Carl H. June ◽  
David Porter ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Humoral Immunity ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric ◽  
Antibody Titers

Abstract Introduction: T cells engineered to express chimeric antigen receptors (CARs) recognizing CD19 (CART19) can eliminate malignant cells in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). We and other groups have shown that persistent tumor eradication by CD19-specific T cell immunotherapy is accompanied by normal B-cell aplasia. It is assumed that responding patients cannot make new antibody responses post-successful CART19 treatment; however, the status of previously established humoral immunity in these patients is currently unknown. Understanding the consequence of successful CART19 therapy on established humoral immunity has implications for both the clinical management of CART19-treated patients as well as the potential application of this therapy to non-malignant diseases such as autoimmunity and transplantation. Methods: We performed a prospective, observational study of adult and pediatric patients with ALL and adults with relapsed/refractory CLL, who were enrolled in clinical trials of CART19 at our institution. Serum antibody titers to previously-generated vaccine or vaccine-related pathogens (Streptococcus pneumoniae, Tetanus toxoid, Hemophilus influenza type-B (HIB), Measles, Mumps, and Rubella) were determined along with a quantitative assessment of B-cell and plasma cell frequencies in blood and bone marrow aspirates. Specimens were collected during pre-established study assessments or additional time points when collected as required for clinical management. Due to the challenges of assessing plasma cells, multiple methods were employed for their quantification in fresh specimens including flow cytometry and immunohistochemistry (IHC). Flow cytometric assessment of plasma cells was performed on freshly obtained marrow samples. Only patients with at least 3 months of B-cell aplasia in the absence of regular intravenous immunoglobulin (IVIg) infusions were included in the study. Results: All patients had no evidence of leukemia or peripheral B cells post-CART19 infusion at the time of this study. Compared to pre-CART19 serum titers, antibodies to S. pneumoniae remained stable or increased in 9 of 12 patients despite lack of circulating B-cells. Antibody titers to Tetanus toxoid were stable or increased in 13 of 14 patients. Anti-HIB levels were stable or increased in 9 of 11 patients and antibodies to Measles, Mumps and Rubella were stable or increased in 12 of 13, 11 of 13, and 12 of 13 patients, respectively. Flow cytometric analysis of bone marrow aspirates after CART19 infusion revealed three patients with persistence of CD38+ CD138+ plasma cells (at 1, 3 and 9 months post infusion, respectively) despite a complete absence of peripheral CD19+ B cells. In 9 patients, CD20 and CD138 IHC analysis of bone marrow core biopsies revealed a decrease in plasma cell (ranges: 1-5% pre-CART19, 0-<1% post-CART19), consistent with our previously published data. Finally, in another subset of patients, neither B cells nor plasma cells were detectable by flow cytometry of aspirate material or IHC of core biopsies collected either pre- or post-CART19 treatment. Conclusions: The stable or increased titers of antibodies to previous vaccines are surprising and may, in part, reflect improved marrow function as a result of leukemia eradication. The demonstration of plasma cells in a subset of patients in the absence of detectable tumor or normal B cells provides strong evidence for the existence of a population of plasma cells that are resistant to lysis by CART19 cells. This is consistent with antibody titers to previously generated vaccine antigens, which remain stable despite effective CART19 treatment. The additional finding of a decrease in CD138+ cells in several patients by IHC suggests that some populations of plasma cells are either targeted directly by CART19 or have a short half-life (e.g. plasmablasts); CD138 is not sufficient to distinguish these populations. Overall, these results indicate that long-lived plasma cells are resistant to CART19, likely due to a loss of CD19 during plasma cell differentiation. Continued analysis of remaining plasma cells in the absence of ongoing B-cell maturation as a result of CART19 persistence may provide important information on turnover rates of these long-lived cells in humans. Disclosures Bhoj: Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties. Porter:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Lacey:Novartis: Research Funding. Mahnke:Novartis: Research Funding.

Rapid and Sensitive Detection of Minimal Plasma Cell Populations Using Six-Colour Flow Cytometry.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3526-3526
Author(s):  
Ruth M. de Tute ◽  
Selina J. Denman ◽  
J. Anthony Child ◽  
Roger G. Owen ◽  
Andy C. Rawstron
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Limit Of Detection ◽  
Cell Phenotype ◽  
High Dose

Abstract Flow cytometry is a powerful tool for diagnosis and detection of minimal residual disease (MRD) in patients with monoclonal gammopathies. Multi-parameter MRD flow analysis is more sensitive than serum immunofixation for the detection of residual disease and is as effective as PCR analysis for predicting outcome after high-dose therapy. Attaining a limit of detection of 0.01% abnormal plasma cells in every patient requires analysis of several neoplastic markers. However, basic immunophenotyping coupled with clonality assessment can be informative in many patients. This approach can also be helpful at diagnosis in MGUS patients to determine whether the primary abnormal component is plasmacytoid or lymphoid. The aim of this study was to develop a single six-colour assay that could be used as a rapid and sensitive screen for the presence of neoplastic plasma cells. Bone marrow aspirate samples were assessed from 83 patients at diagnosis with a suspected plasma cell disorder (median age 69.1 years, 48 male and 35 female) and from 62 patients with myeloma after treatment (median age 61.0 years, 40 male and 22 female). Cells were washed and surface-labelled with CD19 PE, CD38 PE-Cy7, CD138 APC and CD45 APC-Cy7 then fixed, permeabilised and incubated with Kappa FITC and Lambda PE-Cy5. The assay allows assessment of B-cell surface-Ig and plasma cell cytoplasmic-Ig light chain restriction and abnormal plasma cell phenotype according to CD19 and CD45 expression. In presentation cases with more than 10% plasma cells by morphology, neoplastic plasma cells were present in 48/50 samples with one reactive plasmacytosis (32% plasma cells) and one patient with Castlemans disease (11% PC). In the remaining 33 cases with <10% plasma cells, 16/33 (48%) had monoclonal plasma cells, 4/33 (12.1%) showed a low-level of monoclonal B-cells and the remainder had normal B-cells and plasma cells. The assay provided a quantitative MRD result in 44/62 (70%) cases: neoplastic plasma cells represented <1% of leucocytes in 26/44 of the samples (59%) and the assay could detect neoplastic plasma cells when they represented as few as 0.01% of total leucocytes in several patients (4/44, 9.1%). Using this screening approach an extended MRD analysis panel was only required in 18/62 (30%) of cases where CD19- plasma cells were detected on a polyclonal background. Marrow quality was compared with a paired trephine biopsy in 68 samples. Using a algorithm based on the levels of normal plasma cells, B-progenitors, nucleated red cells and myeloid progenitors it was possible to determine whether the samples were representative bone marrow and a quantitative result could be provided. This single tube six-colour approach allows simultaneous assessment of B-cell and plasma cell immunophenotype and clonality. The incorporation of all the relevant markers into a single test makes analysis more straightforward and the gating strategy more reproducible. The assay is more rapid and cost-effective than four-colour analysis as fewer total markers are required. It can be applied to presentation samples to exclude reactive plasmacytoses and B-lymphoproliferative disorders, and to follow up samples to reduce the need for further flow cytometric investigations and optimise any downstream testing that is necessary. In approximately 70% of post-treatment myeloma samples this assay is capable of providing a definitive MRD result up to a maximum sensitivity of 0.01%.

Trogocytosis of multiple B-cell surface markers by CD22 targeting with epratuzumab

Blood ◽  
2013 ◽  
Vol 122 (17) ◽  
pp. 3020-3029 ◽  
Author(s):  
Edmund A. Rossi ◽  
David M. Goldenberg ◽  
Rosana Michel ◽  
Diane L. Rossi ◽  
Daniel J. Wallace ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
Cell Surface ◽  
B Cell ◽  
Regulatory Proteins ◽  
Surface Markers ◽  
B Cell Antigen Receptor ◽  
Cell Surface Markers ◽  
Effector Cells ◽  
Key Points

Key Points Epratuzumab induces the reduction of multiple B-cell antigen receptor–modulating proteins on the surface of B cells via their trogocytosis to effector cells. Modulation of B cells by trogocytosis of key regulatory proteins may be an important mechanism of immunotherapy of autoimmune disease.

scholarly journals Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kristian Assing ◽  
Christian Nielsen ◽  
Marianne Jakobsen ◽  
Charlotte B. Andersen ◽  
Kristin Skogstrand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Formation ◽  
Memory B Cells ◽  
Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

scholarly journals Complement receptors regulate differentiation of bone marrow plasma cell precursors expressing transcription factors Blimp-1 and XBP-1

2005 ◽  
Vol 201 (6) ◽  
pp. 993-1005 ◽  
Author(s):  
Dominique Gatto ◽  
Thomas Pfister ◽  
Andrea Jegerlehner ◽  
Stephen W. Martin ◽  
Manfred Kopf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Cell Activation ◽  
Plasma Cells ◽  
Antibody Responses ◽  
Complement Receptors ◽  
Essentially Normal ◽  
Bone Marrow Plasma

Humoral immune responses are thought to be enhanced by complement-mediated recruitment of the CD21–CD19–CD81 coreceptor complex into the B cell antigen receptor (BCR) complex, which lowers the threshold of B cell activation and increases the survival and proliferative capacity of responding B cells. To investigate the role of the CD21–CD35 complement receptors in the generation of B cell memory, we analyzed the response against viral particles derived from the bacteriophage Qβ in mice deficient in CD21–CD35 (Cr2−/−). Despite highly efficient induction of early antibody responses and germinal center (GC) reactions to immunization with Qβ, Cr2−/− mice exhibited impaired antibody persistence paralleled by a strongly reduced development of bone marrow plasma cells. Surprisingly, antigen-specific memory B cells were essentially normal in these mice. In the absence of CD21-mediated costimulation, Qβ-specific post-GC B cells failed to induce the transcriptional regulators Blimp-1 and XBP-1 driving plasma cell differentiation, and the antiapoptotic protein Bcl-2, which resulted in failure to generate the precursor population of long-lived plasma cells residing in the bone marrow. These results suggest that complement receptors maintain antibody responses by delivery of differentiation and survival signals to precursors of bone marrow plasma cells.

The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-5
Author(s):  
Ling Tian ◽  
Monique Chavez ◽  
Lukas D Wartman
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Development ◽  
Young Female ◽  
B Cell Development ◽  
Cell Immune Response ◽  
Germinal Center Hyperplasia

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2321-2326 ◽  
Author(s):  
D van der Harst ◽  
D de Jong ◽  
J Limpens ◽  
PM Kluin ◽  
Y Rozier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Malignant Lymphoma ◽  
Idiopathic Thrombocytopenic Purpura ◽  
Clinical Manifestations ◽  
Cell Populations ◽  
Dna Rearrangement ◽  
Thrombocytopenic Purpura

Idiopathic thrombocytopenic purpura (ITP) may be associated with other autoimmune diseases and the development of lymphoproliferative malignancies. In Sjogren's disease, Graves' disease, and essential mixed cryoglobulinemia, which are also associated with the development of B-cell neoplasia, clonal B-cell expansions have been detected. Eleven patients with ITP were investigated for the presence of a clonal excess (CE) using kappa-lambda flow cytometry and DNA analysis for rearrangement of immunoglobulin heavy and light chain genes in blood and/or spleen lymphocytes. In 10 of 11 patients, clonal B-cell populations were found by one or both tests. In three of these patients, oligoclonal B-cell populations were suggested by the combined findings. In all four patients with a small paraproteinemia, the isotype was confirmed by either flow cytometry or DNA rearrangement analysis. Our data suggest that the oligoclonal expansions are not restricted to CD5+ B cells, as in the majority of patients this subset was below the detection level of flow cytometry or DNA rearrangement analysis. None of the patients developed clinical manifestations of malignant lymphoma during a follow-up period of 10 to 44 months after sampling. We conclude that clonal excess populations of B cells are not a unique feature of malignant lymphoma, but may occur in autoimmune diseases, suggesting a benign (oligo)clonal B-cell proliferation.

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 Multiscale Modeling of Germinal Center Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells Differentiation as Regulated by Antigen Affinity-Based Tfh Cell Help

2021 ◽  
Vol 11 ◽  
Author(s):  
Elena Merino Tejero ◽  
Danial Lashgari ◽  
Rodrigo García-Valiente ◽  
Xuefeng Gao ◽  
Fabien Crauste ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Asymmetric Division ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Germinal Center Reaction

Germinal centers play a key role in the adaptive immune system since they are able to produce memory B cells and plasma cells that produce high affinity antibodies for an effective immune protection. The mechanisms underlying cell-fate decisions are not well understood but asymmetric division of antigen, B-cell receptor affinity, interactions between B-cells and T follicular helper cells (triggering CD40 signaling), and regulatory interactions of transcription factors have all been proposed to play a role. In addition, a temporal switch from memory B-cell to plasma cell differentiation during the germinal center reaction has been shown. To investigate if antigen affinity-based Tfh cell help recapitulates the temporal switch we implemented a multiscale model that integrates cellular interactions with a core gene regulatory network comprising BCL6, IRF4, and BLIMP1. Using this model we show that affinity-based CD40 signaling in combination with asymmetric division of B-cells result in switch from memory B-cell to plasma cell generation during the course of the germinal center reaction. We also show that cell fate division is unlikely to be (solely) based on asymmetric division of Ag but that BLIMP1 is a more important factor. Altogether, our model enables to test the influence of molecular modulations of the CD40 signaling pathway on the production of germinal center output cells.

scholarly journals The suppression of Brd4 inhibits peripheral plasma cell differentiation and exhibits therapeutic potential for systemic lupus erythematosus

2021 ◽  
Author(s):  
Shan Zeng ◽  
Qian Qiu ◽  
Yi Zhou ◽  
Youjun Xiao ◽  
Jingnan Wang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Lupus Erythematosus ◽  
Plasma Cells ◽  
Therapeutic Potential ◽  
Systemic Lupus ◽  
Plasma Cell Differentiation ◽  
Brd4 Inhibition

Background and purpose: To investigate the role of bromodomain-containing protein 4 (Brd4) in regulating B cell differentiation and its therapeutic potential for B cell-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). Experimental Approach: Human and murine B cells were purified and cultured with different stimuli. B cell surface markers, proliferation and apoptosis were estimated by flow cytometry. Gene expression was measured by quantitative real-time PCR. Brd4 binding sites were analysed by the luciferase reporter assay and the chromatin immunoprecipitation (ChIP) assay. PFI-1 or JQ1 was used to inhibit Brd4. Mice with B cell-specific deletion of the Brd4 gene (Brd4flox/floxCD19-Cre+/-) and MRL/lpr mice were used to perform the in vivo experiments. Key Results: Brd4 inhibition suppressed plasmablast-mediated plasma cell differentiation but did not influence proliferation or apoptosis in healthy human and murine CD19+ B cells. PFI-1 treatment reduced the secretion of IgG and IgM in the supernatants of costimulation-induced B cells. Mechanistically, Brd4 regulates the terminal differentiation of B cells into plasma cells by targeting BLIMP1 by directly binding and activating the endogenous BLIMP1 promoter. Interestingly, PFI-1 treatment decreased the percentages of plasmablasts and plasma cells from patients with SLE. PFI-1 administration reduced the percentages of plasma cells, hypergammaglobulinemia and attenuated nephritis in MRL/lpr mice. Pristane-injected Brd4flox/floxCD19-Cre+/- mice exhibited improved nephritis and reduced percentages of plasma cells. Conclusions and Implications: Brd4 is an essential factor in regulating plasma cell differentiation. Brd4 inhibition may be a potential new strategy for the treatment of B cell-associated autoimmune disorders, including SLE.

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