scholarly journals Developmental Switches in Chemokine Response Profiles during B Cell Differentiation and Maturation

2000 ◽  
Vol 191 (8) ◽  
pp. 1303-1318 ◽  
Author(s):  
Edward P. Bowman ◽  
James J. Campbell ◽  
Dulce Soler ◽  
Zengjun Dong ◽  
Natasha Manlongat ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Cell Subset ◽  
Lymphoid Tissues ◽  
B Cell Differentiation ◽  
Interleukin 7 ◽  
Developmental Switches

Developing B cells undergo dramatic changes in their responses to chemoattractant cytokines (chemokines) and in expression of chemokine receptors. Bone marrow pre–pro-B cells (AA4.1+/natural killer 1.1− Fraction A cells) and cells capable of generating pro-B colonies in the presence of interleukin 7 and flt3 ligand migrate to thymus-expressed chemokine (TECK), a response lost in later stages of B cell development. B cell–attracting chemokine 1 (BCA-1) responses correlate with CXC chemokine receptor (CXCR)5 expression, are first displayed by a pro-B cell subset, are lost in pre-B cells, and then are regained just before and after egress from the marrow. All peripheral B cell subsets, including follicular and germinal center as well as marginal zone and peritoneal B1 B cells, respond to BCA-1, implying that responsiveness to this follicular chemokine is not sufficient to predict follicle localization. Responses to the CC chemokine receptor (CCR)7 ligands secondary lymphoid tissue chemoattractant (SLC) and macrophage inflammatory protein (MIP)-3β, implicated in homing to lymphoid tissues, are upregulated before B cell exit from the marrow, but increase further in the periphery and are shared by all peripheral B cells. In contrast, responsiveness to MIP-3α and expression of CCR6 are acquired only after emigration to the periphery and during maturation into the recirculating B cell pool. Chemotaxis to stromal cell–derived factor 1α is observed at all stages of B cell differentiation. Thus, unique patterns of chemokine responses may help define developing B cell populations and direct their maturation in the marrow and migration to the periphery.

Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3α/CCL20 in human B cells

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2338-2345 ◽  
Author(s):  
Roman Krzysiek ◽  
Eric A. Lefevre ◽  
Jérôme Bernard ◽  
Arnaud Foussat ◽  
Pierre Galanaud ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Chemokine Receptor ◽  
Memory B Cells ◽  
Receptor Expression ◽  
B Cell Differentiation ◽  
Hematopoietic Stem ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract The regulation of CCR6 (chemokine receptor 6) expression during B-cell ontogeny and antigen-driven B-cell differentiation was analyzed. None of the CD34+Lin− hematopoietic stem cell progenitors or the CD34+CD19+ (pro-B) or the CD19+CD10+ (pre-B/immature B cells) B-cell progenitors expressed CCR6. CCR6 is acquired when CD10 is lost and B-cell progeny matures, entering into the surface immunoglobulin D+ (sIgD+) mature B-cell pool. CCR6 is expressed by all bone marrow–, umbilical cord blood–, and peripheral blood–derived naive and/or memory B cells but is absent from germinal center (GC) B cells of secondary lymphoid organs. CCR6 is down-regulated after B-cell antigen receptor triggering and remains absent during differentiation into immunoglobulin-secreting plasma cells, whereas it is reacquired at the stage of post-GC memory B cells. Thus, within the B-cell compartment, CCR6 expression is restricted to functionally mature cells capable of responding to antigen challenge. In transmigration chemotactic assays, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20 (CCL20) induced vigorous migration of B cells with differential chemotactic preference toward sIgD− memory B cells. These data suggest that restricted patterns of CCR6 expression and MIP-3α/CCL20 responsiveness are integral parts of the process of B-lineage maturation and antigen-driven B-cell differentiation.

CCL19 Induced Responses Are Differentially Regulated by Atypical Chemokine Receptor CRAM and Its Classical Chemokine Receptor CCR7 in B-CLL Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4896-4896 ◽  
Author(s):  
Nathalie Burger ◽  
Andrea Haerzschel ◽  
Marion Leick ◽  
Tanja Nicole Hartmann ◽  
Julie Catusse ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Conflicts Of Interest ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Dependent Manner ◽  
Chemokine Ligand ◽  
Cell Layers

Abstract Abstract 4896 Introduction: Chemokines are known to play an important role in the migration and survival of B-CLL cells. The non-signalling chemokine receptors, including DARC, D6 and CCX-CKR, have recently been shown to be involved in chemokine clearance and activity regulation. The human chemokine receptor CRAM is the most recently identified member of this atypical group. CRAM is expressed on B cells in a maturation-stage dependent manner, and to variable degrees on B-CLL cells. We have recently shown that it competitively binds CCL19 and that this binding is not followed by classical chemokine responses. CCL19 and its signalling receptor CCR7 are centrally involved in B cell localisation and maturation within the secondary lymphoid tissues. CCR7 is also highly expressed on B cells from CLL patients and mediates migration towards its ligands CCL19 and CCL21 which have been shown to be present at higher concentrations in serum of patients with lymphadenopathia compared to patients without. In this study we investigate the influence of CRAM on the CCL19 dependent responses of B-CLL cells and potential correlations to clinical data with a specific focus on lymphadenopathia. Results: We demonstrate that B cells from patients with B-CLL present high, but variable degrees of CCR7 and CRAM expression. Patients with compared to patients without lymphadenopathia show a higher CRAM expression level whereas the CCR7 expression is not significantly different. In single samples showing extremly high CRAM expression the migration towards CCL19 is reduced compared to patients with lower CRAM expression. These observations confirm results in the B-CLL cell line MEC-1 showing increased migration toward CCL19 when CRAM expression is reduced using CRAM-siRNA. On the other hand, CRAM seems to be a chemokine presenter as we can show that it does not degrade its chemokine ligand but presents it on the surface of polarised cell layers. Thus, we assume that CRAM plays a role for cell migration, possibly transmigration and cell localisation within lymph nodes of B-CLL cells. Conclusions: We show that CRAM can act as an integrator of different recruitment and activation factors. It is associated to CCR7 driven recruitment of B cells by regulating CCL19 availability. Expression of CRAM differs in B cell malignancies for which CCL19 and CCL21 have already been shown to be implicated in lymphadenopathia. We therefore suggest that CRAM is an additional player in the localisation and differentiation/maturation processes of malignant B cells of B-CLL patients. Disclosures: No relevant conflicts of interest to declare.

Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3α/CCL20 in human B cells

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2338-2345 ◽  
Author(s):  
Roman Krzysiek ◽  
Eric A. Lefevre ◽  
Jérôme Bernard ◽  
Arnaud Foussat ◽  
Pierre Galanaud ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Chemokine Receptor ◽  
Memory B Cells ◽  
Receptor Expression ◽  
B Cell Differentiation ◽  
Hematopoietic Stem ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

The regulation of CCR6 (chemokine receptor 6) expression during B-cell ontogeny and antigen-driven B-cell differentiation was analyzed. None of the CD34+Lin− hematopoietic stem cell progenitors or the CD34+CD19+ (pro-B) or the CD19+CD10+ (pre-B/immature B cells) B-cell progenitors expressed CCR6. CCR6 is acquired when CD10 is lost and B-cell progeny matures, entering into the surface immunoglobulin D+ (sIgD+) mature B-cell pool. CCR6 is expressed by all bone marrow–, umbilical cord blood–, and peripheral blood–derived naive and/or memory B cells but is absent from germinal center (GC) B cells of secondary lymphoid organs. CCR6 is down-regulated after B-cell antigen receptor triggering and remains absent during differentiation into immunoglobulin-secreting plasma cells, whereas it is reacquired at the stage of post-GC memory B cells. Thus, within the B-cell compartment, CCR6 expression is restricted to functionally mature cells capable of responding to antigen challenge. In transmigration chemotactic assays, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20 (CCL20) induced vigorous migration of B cells with differential chemotactic preference toward sIgD− memory B cells. These data suggest that restricted patterns of CCR6 expression and MIP-3α/CCL20 responsiveness are integral parts of the process of B-lineage maturation and antigen-driven B-cell differentiation.

scholarly journals THU0043 EXPRESSION OF B CELL CHEMOKINE-CHEMOKINE RECEPTOR PATHWAYS IS ALTERED IN GIANT CELL ARTERITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 234.2-234
Author(s):  
J. Graver ◽  
A. Boots ◽  
W. Abdulahad ◽  
J. Bijzet ◽  
D. Wolbers ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Chemokine Receptor ◽  
Temporal Artery ◽  
Absolute Number ◽  
Memory B Cells ◽  
Receptor Expression ◽  
Healthy Controls ◽  
B Cell Differentiation

Background:The presence of organised B cells in both cranial-giant cell arteritis (C-GCA) (temporal artery) and large vessel (LV)-GCA (aorta) has previously been documented. The number and the extent of organisation of B cells in tertiary lymphoid organs (TLO) was more prominent in the aorta than in the temporal artery, suggesting possible differences in B cell phenotype, kinetics and tropism between C-GCA and LV-GCA.Objectives:We sought to analyse B cell differentiation subsets in both C-GCA and LV-GCA and to investigate differences in the expression of chemokine pathways involved in B cell migration and TLO organisation.Methods:Blood was collected from C-GCA (n=11) and LV-GCA (n=22) patients at baseline, before start of glucocorticoid treatment, and after 3 months of treatment. The LV-GCA groups consisted of 11 patients with isolated LV-GCA and 11 patients with overlap LV/C-GCA. Also, age- and sex- matched healthy controls (HC, n=24) were included. The following chemokines were measured with Luminex in the sera of patients and HC: BAFF, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL12, and CXCL13. Thawed PBMC of 7 C-GCA, 10 LV-GCA and 24 HC were stained with antibodies against CD19, CD27, IgD, IgM, CD38, CXCR3, CXCR4, CXCR5, and CCR7 to allow identification of B cell differentiation subsets and their chemokine receptor expression.Results:We found a lower absolute number of CXCR3+ memory and double negative (late stage) B cells in GCA patients when compared to healthy controls. Also, the absolute number of CXCR5+ memory B cells was lower in patients than in controls. Chemokine receptor expression on circulating B cells did not significantly differ between C-GCA and LV-GCA at baseline. After 3 months of treatment, frequencies and absolute numbers of both CXCR3+ and CXCR5+ memory B cells increased. In sera of all GCA patients, CXCL9 (which is a chemokine involved in migration of B cells to sites of inflammation) and CXCL13 (which is involved in local organization of B cells) were significantly increased. BAFF and CCL21 were increased only in LV-GCA when compared to HC. Serum chemokine levels did not differ between C-GCA and LV-GCA patients. An inverse correlation was observed between B cell counts and CXCL9 as well as CXCL13 in LV-GCA, only. After 3 months of treatment, CXCL9 levels remained elevated whereas CXCL13 increased even further.Conclusion:At diagnosis, CXCL9 and CXCL13 were significantly increased in all GCA patients as compared to HC. Elevated CXCL9 levels inversely correlated with B cells numbers in LV-GCA, only, which may suggest that B cells preferentially migrate to the inflamed aorta via a mechanism involving CXCL9. In addition, CXCL13 may be linked to local TLO organization in LV-GCA. Currently, we are studying the local expression of chemokines and chemokine receptors at the site of inflammation in both C- and LV-GCA.Disclosure of Interests:Jacoba Graver: None declared, Annemieke Boots Consultant of: Grünenthal Gmbh until 2017, Wayel Abdulahad: None declared, Johan Bijzet: None declared, Daphne Wolbers: None declared, Elisabeth Brouwer Consultant of: Roche (consultancy fee 2017 and 2018 paid to the UMCG), Speakers bureau: Roche (2017 and 2018 paid to the UMCG), Maria Sandovici: None declared

scholarly journals Loss of Bim Allows Precursor B Cell Survival But Not Precursor B Cell Differentiation in the Absence of Interleukin 7

2004 ◽  
Vol 200 (9) ◽  
pp. 1179-1187 ◽  
Author(s):  
Paula M. Oliver ◽  
Michael Wang ◽  
Yanan Zhu ◽  
Janice White ◽  
John Kappler ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
B Cell Differentiation ◽  
Normal Numbers ◽  
Cell Precursor ◽  
Interleukin 7 ◽  
B Cell Survival ◽  
B Cell Precursors

Interleukin (IL)-7 is a stromal cell–derived cytokine required for the survival, proliferation, and differentiation of B cell precursors. Members of the Bcl-2 family of proteins are known to have profound effects on lymphocyte survival, but not lymphocyte differentiation. To distinguish the relative dependence on IL-7 of B cell precursor survival versus B cell differentiation, the combined effects of lack of IL-7 and lack of the proapoptotic Bcl-2 relative, Bim, were studied. Bim is expressed to varying degrees in all B cell precursors and B cells. Lack of Bim compensated for lack of IL-7 in the survival of pro–, pre–, and immature B cells; however, lack of Bim did not substitute for the requirement for IL-7 in B cell precursor differentiation or B cell precursor proliferation. Precursor B cell survival is more dependent on sufficient levels of IL-7 than precursor B cell differentiation because the number of B cells and their precursors were reduced by half in mice heterozygous for IL-7 expression, but were restored to normal numbers in mice also lacking Bim. Hence, Bim and IL-7 work together to control the survival of B cell precursors and the number of B cells that exist in animals.

scholarly journals IL-21 and IFN-α have both opposite and redundant role on human innate precursors and memory B-cell differentiation.

2021 ◽  
Author(s):  
Marina Boudigou ◽  
Magalie Michée-Cospolite ◽  
Patrice Hémon ◽  
Alexis Grasseau ◽  
Christelle Le Dantec ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Memory B Cells ◽  
Lymphoid Tissues ◽  
B Cell Differentiation ◽  
Functional Responses ◽  
Marginal Zone B Cells ◽  
Activated State

Immunological memory is essential for effective immune protection upon antigen rechallenge. Memory B cells encompass multiple subsets, heterogeneous in terms of phenotypes, origins and precursors, anatomical localization, and functional responses. B-cell responses are conditioned by micro-environmental signals, including cytokines. Here, we analyzed in vitro the effects of two cytokines implicated in B-cell differentiation, interferon-alpha (IFN-α) and interleukin (IL)-21, on the early functional response of four different mature B-cell subsets (IgD- CD27- naive, IgD+ CD27+ unswitched, IgD- CD27+ switched and double-negative B cells). The dual response of naive and memory B cells to IL-21 allowed us to uncover a unique IgD+ CD27- CD10- B-cell population (referred to as NARB+) characterized by the expression of marginal zone B-cell markers CD45RB and CD1c. Similar to memory B cells, NARB+ cells were in a pre-activated state, allowing them to rapidly differentiate into plasmablasts upon innate signals while maintaining their susceptibility to IL-21 activation-induced apoptosis as observed for the naive compartment. Both in-depth phenotypic analysis of circulating B cells, and identification of these cells in spleen, tonsil and gut-associated lymphoid tissues, supported that NARB+ are uncommitted precursors of human marginal zone B cells.

scholarly journals Dynamic Intracellular Metabolic Cell Signaling Profiles During Ag-Dependent B-Cell Differentiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Paula Díez ◽  
Martín Pérez-Andrés ◽  
Martin Bøgsted ◽  
Mikel Azkargorta ◽  
Rodrigo García-Valiente ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Metabolic Regulation ◽  
Cell Subset ◽  
Maturation Stage ◽  
Label Free ◽  
B Cell Differentiation ◽  
Cell Subpopulations ◽  
Human B Cell

Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells undergoing antigen-dependent maturation using a label-free LC-MS/MS approach applied on 5 purified B-cell subpopulations (naive, centroblasts, centrocytes, memory and plasma B-cells) from human tonsils (data are available via ProteomeXchange with identifier PXD006191). Our results revealed that the actual differences among these B-cell subpopulations are a combination of expression of a few maturation stage-specific proteins within each B-cell subset and maturation-associated changes in relative protein expression levels, which are related with metabolic regulation. The considerable overlap of the proteome of the 5 studied B-cell subsets strengthens the key role of the regulation of the stoichiometry of molecules associated with metabolic regulation and programming, among other signaling cascades (such as antigen recognition and presentation and cell survival) crucial for the transition between each B-cell maturation stage.

AB0144 STRATIFICATION OF PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME BY MEASURING B-CELL HEALTH

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1372-1373
Author(s):  
G. M. Verstappen ◽  
J. C. Tempany ◽  
H. Cheon ◽  
A. Farchione ◽  
S. Downie-Doyle ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Cell Differentiation ◽  
Research Support ◽  
Differentiation Capacity ◽  
Cell Responses ◽  
Healthy Donors

Background:Primary Sjögren’s syndrome (pSS) is a heterogeneous immune disorder with broad clinical phenotypes that can arise from a large number of genetic, hormonal, and environmental causes. B-cell hyperactivity is considered to be a pathogenic hallmark of pSS. However, whether B-cell hyperactivity in pSS patients is a result of polygenic, B cell-intrinsic factors, extrinsic factors, or both, is unclear. Despite controversies about the efficacy of rituximab, new B-cell targeting therapies are under investigation with promising early results. However, for such therapies to be successful, the etiology of B-cell hyperactivity in pSS needs to be clarified at the individual patient level.Objectives:To measure naïve B-cell function in pSS patients and healthy donors using quantitative immunology.Methods:We have developed standardised, quantitative functional assays of B-cell responses that measure division, death, differentiation and isotype switching, to reveal the innate programming of B cells in response to T-independent and dependent stimuli. This novel pipeline to measure B-cell health was developed to reveal the sum total of polygenic defects and underlying B-cell dysfunction at an individual level. For the current study, 25 pSS patients, fulfilling 2016 ACR-EULAR criteria, and 15 age-and gender-matched healthy donors were recruited. Standardized quantitative assays were used to directly measure B cell division, death and differentiation in response to T cell-independent (anti-Ig + CpG) and T-cell dependent (CD40L + IL-21) stimuli. Naïve B cells (IgD+CD27-) were sorted from peripheral blood mononuclear cells and were labeled with Cell Trace Violet at day 0 to track cell division until day 6. B cell differentiation was measured at day 5.Results:Application of our standardized assays, and accompanying parametric models, allowed us to study B cell-intrinsic defects in pSS patients to a range of stimuli. Strikingly, we demonstrated a hyperresponse of naïve B cells to combined B cell receptor (BCR) and Toll-like receptor (TLR)-9 stimulation in pSS patients. This hyperresponse was revealed by an increased mean division number (MDN) at day 5 in pSS patients compared with healthy donors (p=0.021). A higher MDN in pSS patients was observed at the cohort level and was likely attributed to an increased division burst (division destiny) time. The MDN upon BCR/TLR-9 stimulation correlated with serum IgG levels (rs=0.52; p=0.011). No difference in MDN of naïve B cells after T cell-dependent stimulation was observed between pSS patients and healthy donors. B cell differentiation capacity (e.g., plasmablast formation and isotype switching) after T cell-dependent stimulation was also assessed. At the cohort level, no difference in differentiation capacity between groups was observed, although some pSS patients showed higher plasmablast frequencies than healthy donors.Conclusion:Here, we demonstrate defects in B-cell responses both at the cohort level, as well as individual signatures of defective responses. Personalized profiles of B cell health in pSS patients reveal a group of hyperresponsive patients, specifically to combined BCR/TLR stimulation. These patients may benefit most from B-cell targeted therapies. Future studies will address whether profiles of B cell health might serve additional roles, such as prediction of disease trajectories, and thus accelerate early intervention and access to precision therapies.Disclosure of Interests:Gwenny M. Verstappen: None declared, Jessica Catherine Tempany: None declared, HoChan Cheon: None declared, Anthony Farchione: None declared, Sarah Downie-Doyle: None declared, Maureen Rischmueller Consultant of: Abbvie, Bristol-Meyer-Squibb, Celgene, Glaxo Smith Kline, Hospira, Janssen Cilag, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Ken R. Duffy: None declared, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Philip D. Hodgkin Grant/research support from: Medimmune, Vanessa L. Bryant Grant/research support from: CSL

scholarly journals YY1 plays an essential role at all stages of B-cell differentiation

2016 ◽  
Vol 113 (27) ◽  
pp. E3911-E3920 ◽  
Author(s):  
Eden Kleiman ◽  
Haiqun Jia ◽  
Salvatore Loguercio ◽  
Andrew I. Su ◽  
Ann J. Feeney
Keyword(s):  
Transcription Factor ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Signaling Pathways ◽  
Mrna Splicing ◽  
Cell Populations ◽  
Sequencing Analysis ◽  
B Cell Differentiation ◽  
Chip Sequencing

Ying Yang 1 (YY1) is a ubiquitously expressed transcription factor shown to be essential for pro–B-cell development. However, the role of YY1 in other B-cell populations has never been investigated. Recent bioinformatics analysis data have implicated YY1 in the germinal center (GC) B-cell transcriptional program. In accord with this prediction, we demonstrated that deletion of YY1 by Cγ1-Cre completely prevented differentiation of GC B cells and plasma cells. To determine if YY1 was also required for the differentiation of other B-cell populations, we deleted YY1 with CD19-Cre and found that all peripheral B-cell subsets, including B1 B cells, require YY1 for their differentiation. Transitional 1 (T1) B cells were the most dependent upon YY1, being sensitive to even a half-dosage of YY1 and also to short-term YY1 deletion by tamoxifen-induced Cre. We show that YY1 exerts its effects, in part, by promoting B-cell survival and proliferation. ChIP-sequencing shows that YY1 predominantly binds to promoters, and pathway analysis of the genes that bind YY1 show enrichment in ribosomal functions, mitochondrial functions such as bioenergetics, and functions related to transcription such as mRNA splicing. By RNA-sequencing analysis of differentially expressed genes, we demonstrated that YY1 normally activates genes involved in mitochondrial bioenergetics, whereas it normally down-regulates genes involved in transcription, mRNA splicing, NF-κB signaling pathways, the AP-1 transcription factor network, chromatin remodeling, cytokine signaling pathways, cell adhesion, and cell proliferation. Our results show the crucial role that YY1 plays in regulating broad general processes throughout all stages of B-cell differentiation.

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.

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