Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells

Engagement of cytokine receptors by specific ligands activate Janus kinase–signal transducer and activator of transcription (STAT) signaling pathways. The exact roles of STATs in human lymphocyte behavior remain incompletely defined. Interleukin (IL)-21 activates STAT1 and STAT3 and has emerged as a potent regulator of B cell differentiation. We have studied patients with inactivating mutations in STAT1 or STAT3 to dissect their contribution to B cell function in vivo and in response to IL-21 in vitro. STAT3 mutations dramatically reduced the number of functional, antigen (Ag)-specific memory B cells and abolished the ability of IL-21 to induce naive B cells to differentiate into plasma cells (PCs). This resulted from impaired activation of the molecular machinery required for PC generation. In contrast, STAT1 deficiency had no effect on memory B cell formation in vivo or IL-21–induced immunoglobulin secretion in vitro. Thus, STAT3 plays a critical role in generating effector B cells from naive precursors in humans. STAT3-activating cytokines such as IL-21 thus underpin Ag-specific humoral immune responses and provide a mechanism for the functional antibody deficit in STAT3-deficient patients.

Download Full-text

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

10.20944/preprints202111.0365.v1 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood-2008-10-185421 ◽

2009 ◽

Vol 113 (17) ◽

pp. 3969-3977 ◽

Cited By ~ 102

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.

Download Full-text

Absence of surface IgD does not impair naive B cell homeostasis and memory B cell formation in humans

10.1101/332361 ◽

2018 ◽

Author(s):

J. Nechvatalova ◽

S.J.W. Bartol ◽

Z. Chovancova ◽

L. Boon ◽

M. Vlkova ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Activation ◽

Genetic Defect ◽

Memory B Cells ◽

Cell Homeostasis ◽

Human B Cells ◽

B Cell Homeostasis

One Sentence SummaryHuman B cells with a genetic defect in IGHD develop normally in vivo, and do not have a competitive disadvantage to IgD-expressing B cells for developing into memory B cells.AbstractSurface immunoglobulin D (IgD) is co-expressed with IgM on naive mature B cells. Still, the role of surface IgD remains enigmatic even 50 years after its initial discovery. We here examined the in vivo role of surface IgD in human B-cell homeostasis and antibody responses in four individuals with heterozygous nonsense mutations in IGHD. All IGHD heterozygous individuals had normal numbers of B cells and serum immunoglobulins, and did not show signs of immunodeficiency or immune dysregulation. IgD+ and IgD– naive mature B cells were present in equal numbers and showed similar immunophenotypes, except for decreased expression of CD79b in the IgD– subset. Furthermore, both IgD+ and IgD– naive mature B cells had normal replication histories, similar capacities to differentiate into plasma cells upon in vitro stimulation, and Ig switched memory B cells showed similar levels of somatic hypermutations. Thus human B cells lacking IgD expression develop normally and generate immunological memory in vivo, suggesting that surface IgD might function more restricted in regulating of B-cell activation to specific antigenic structures.

Download Full-text

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

Journal of Experimental Medicine ◽

10.1084/jem.186.6.931 ◽

1997 ◽

Vol 186 (6) ◽

pp. 931-940 ◽

Cited By ~ 117

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.

Download Full-text

Human B Cells Immortalized with Epstein-Barr Virus Upregulate CCR6 and CCR10 and Downregulate CXCR4 and CXCR5

Journal of Virology ◽

10.1128/jvi.76.6.3072-3077.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 3072-3077 ◽

Cited By ~ 54

Author(s):

Takashi Nakayama ◽

Ryuichi Fujisawa ◽

Dai Izawa ◽

Kunio Hieshima ◽

Kenzo Takada ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Receptor Expression ◽

Stable Expression ◽

B Cell Differentiation ◽

Barr Virus ◽

Human B Cells ◽

Epstein Barr

ABSTRACT Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.

Download Full-text

Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation

10.1101/826370 ◽

2019 ◽

Cited By ~ 1

Author(s):

Muhammad Assad Aslam ◽

Mir Farshid Alemdehy ◽

Eliza Mari Kwesi-Maliepaard ◽

Marieta Caganova ◽

Iris N. Pardieck ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

Plasma Cell ◽

Plasma Cells ◽

B Cell Differentiation ◽

Humoral Immune

AbstractDifferentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B cell fate remain unclear. Here we identified a central role for the histone H3K79 methyltransferase DOT1L in controlling B cell differentiation. Murine B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells showed aberrant differentiation and prematurely acquired plasma cell features. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L supports the repression of an anti-proliferative, plasma cell differentiation program by maintaining expression of the H3K27 methyltransferase Ezh2, the catalytic component of Polycomb Repressor Complex 2 (PRC2). Our findings show that DOT1L is a central modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B cell naivety and GC B cell differentiation.

Download Full-text

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

10.20944/preprints202111.0365.v2 ◽

2021 ◽

Author(s):

Casper Marsman ◽

Dorit Verhoeven ◽

Jana Koers ◽

Theo Rispens ◽

Anja ten Brinke ◽

...

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.

Download Full-text

Bone Morphogenetic Proteins Inhibit CD40L- and IL-21-Induced Ig Production In Human B Cells: Differential Effects of BMP-6 and BMP-7

Blood ◽

10.1182/blood.v116.21.1745.1745 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1745-1745

Author(s):

Kanutte Huse ◽

Maren Bakkeb∅ ◽

Lise Forfang ◽

Vera I. Hilden ◽

Erlend B. Smeland ◽

...

Keyword(s):

Cell Death ◽

Transcription Factors ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Bone Morphogenetic Proteins ◽

Peripheral Blood ◽

Plasma Cells ◽

Memory B Cells ◽

Human B Cells

Abstract Abstract 1745 Introduction: TGF-β has been shown to play an important role in directing class switch recombination in human B cells. Bone morphogenetic proteins (BMPs) are members of the TGF-β family, and we have previously shown that BMP-6 inhibits proliferation of B cell progenitors as well as mature B cells in humans. However, how various BMP members affect Ig production and B-cell maturation to plasma cells have not been investigated. Here we studied the effects of various BMPs in CD27- naive and CD27+ memory B cells from peripheral blood of healthy human donors. Methods: We used flow immunomagnetic beads and flow cytometry cell sorting to purify CD19+CD27- naive and CD19+CD27+ memory B cells from peripheral blood of healthy donors, and investigated BMP induced effects on in vitro proliferation, cell death and Ig production as measured by thymidine incorporation assay, propidium iodide staining and ELISA, respectively. To separate direct inhibition of plasma cell differentiation from indirect effects via suppression of proliferation and induction of apoptosis, CFSE tracking of cell division was combined with immunophenotyping of cultured cells to identify CD27+CD38+ plasma cells. Expression of transcription factors and AICDA were measured by quantitative real-time PCR. Results: BMP-2, -4, -6 and -7 specifically inhibited CD40L- and IL-21-induced production of IgM, IgG and IgA. BMP-6 was the most potent inhibitor, reducing the Ig production by 70% in memory B cells and more than 50% in naive B cells. By investigating the mechanisms for reduced Ig production, we found a striking difference between the structurally similar BMP-6 and BMP-7. BMP-6 directly inhibited differentiation to CD27+CD38+ plasma cells, whereas BMP-7 only had minor effects on differentiation. Instead, BMP-7 mainly affected Ig production indirectly by inducing apoptosis. Furthermore, we explored BMP-6-induced signaling and gene regulation in more detail in memory B cells. BMP-6 up-regulated Id1, Id2 and Id3 gene expression in CD40L- and IL-21-stimulated cells (6.1-fold, 1.7-fold and 4.0-fold induction after 48 hours culture with BMP-6, respectively). In contrast, BMP-6 potently inhibited CD40L- and IL-21-induced upregulation of the transcription factor XBP-1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected, indicating that BMP-6 only modulates late events of plasma cell development. Conclusion: These results show that BMPs potently suppress Ig production in mature human B cells by inhibiting differentiation or by inducing cell death. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CD40 and IgE: synergism between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells.

Journal of Experimental Medicine ◽

10.1084/jem.172.6.1861 ◽

1990 ◽

Vol 172 (6) ◽

pp. 1861-1864 ◽

Cited By ~ 310

Author(s):

H H Jabara ◽

S M Fu ◽

R S Geha ◽

D Vercelli

Keyword(s):

Monoclonal Antibody ◽

B Cells ◽

B Cell ◽

Interleukin 4 ◽

B Cell Differentiation ◽

Human B Cells ◽

Molecular Events ◽

Ige Synthesis ◽

Ige Response

A novel pathway of IgE-B cell differentiation has been identified. Engagement of the B cell antigen CD40 by F(ab')2 fragments of monoclonal antibody (mAb) 626.1 in the presence of recombinant interleukin 4 (rIL-4) induced intense IgE synthesis, but modest IgG synthesis, by highly purified human B cells. Surface IgE- B cells isolated by cell sorting were induced to produce IgE by mAb 626.1 and IL-4. Thus, IgE synthesis is unlikely to result from expansion of a B cell population precommitted to IgE in vivo. A neutralizing anti-IL-6 antibody strongly, but not completely, inhibited the IgE response. This indicates that autocrine production of IL-6 plays an important amplification role in IgE synthesis triggered by anti-CD40 mAb and IL-4. Although the exact role played by CD40 in IgE responses in vivo remains to be established, this T cell-independent system represents a useful model to characterize the biochemical and molecular events leading to IgE synthesis in human B cells.

Download Full-text