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.

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.

The CD21low B Cell Population Is Composed of an Anergic and an Immunostimulatory Germinal Center-like B Cell Subset

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1425-1425
Author(s):  
Alexander Shimabukuro-Vornhagen ◽  
María García Márquez ◽  
Rieke Fischer ◽  
Kerstin Wennhold ◽  
Juliane Iltgen ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Functional State ◽  
Cell Subset ◽  
Cell Compartment ◽  
Inflammatory Conditions ◽  
Cell Subpopulations ◽  
B Cell Subsets ◽  
Antigen Presenting ◽  
Human B Cell

Abstract In recent years we have gained an increased understanding of the complexity of B cell biology and function. It has become increasingly recognized that apart from antibody production B cells exert many more function. B cells serve as antigen-presenting cells (APC), they contribute to immunoregulation and represent an important source of cytokines and chemokines. A deeper understanding of the role of B cells in the pathophysiology of human diseases has been hampered by the lack of well-defined functional B cell subsets. We therefore aimed to identify novel human B cell subsets which could serve as biomarkers or targets of therapeutic intervention. Using a transcriptomic approach combined with flowcytometric immune assessment of healthy human subjects and patients we were able to identify several functional B cell subpopulations with relevance to human disease. We were able to define a CD21low CD86pos human B cell subset with strong antigen-presenting capacity which gradually develops from conventional resting B cells under the continuous stimulation via CD40. These cells were phenotypically and functionally distinct from CD21low CD86neg B lymphocytes, which represent anergic B cells. Using calcium flux assays and phospho-specific flow cytometry we were able to show that the CD21low B cell subsets displayed distinct signaling states. Both CD21low B cell subpopulations had an impaired response to B cell receptor stimulation. However, CD21low CD86pos B cells had higher basal calcium levels and basal phosphorylation of BCR-associated signaling molecules such as Syk and Erk. Contrary to CD21low CD86neg B cells, which demonstrated poor antigen-presenting capacity, CD21low CD86pos B cells were potent immunostimulatory antigen-presenting cells. CD21low CD86pos B cells were increased in acute inflammation and autoimmune diseases such as rheumatoid arthritis. CD21low CD86neg B cells, on the other hand, were increased in chronic inflammatory conditions such as chronic HIV infection. The balance between the CD21low B cell subsets varied with the functional state of the B cell compartment in inflammatory conditions and could be used to classify the functional state of the B cell compartment. In summary, we have identified several novel human B cell subsets with distinct functions. Given the large number of B cell-directed drugs which are in clinical development or already approved it seems likely that an increased knowledge of the human B cell subsets will not only provide important insights into the pathology of immune-mediated diseases but will also result in novel therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals G protein subunit G alpha 16 expression is restricted to progenitor B cells during human B-cell differentiation

Blood ◽  
1995 ◽  
Vol 85 (7) ◽  
pp. 1836-1842 ◽  
Author(s):  
MY Mapara ◽  
K Bommert ◽  
RC Bargou ◽  
C Leng ◽  
C Beck ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Cell Phenotype ◽  
Low Grade ◽  
B Cell Differentiation ◽  
Human B Cell

Recently G alpha 16, a new guanosine triphosphate (GTP) binding protein alpha subunit has been described to be specifically expressed in human hematopoietic cells. Expression of G alpha 16 was observed in human cell lines of myelomonocytic and T-lymphocytic origin, but not in human B-cell lines Raji and IM9. We studied the expression of G alpha 16 in human B cells corresponding to different stages of B-cell differentiation by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The human Burkitt's lymphoma cell lines Raji, Ramos, BJAB, the lymphoblastoid cell line SKW6.4, and the plasmocytoma cell line U266 were devoid of G alpha 16. In contrast, G alpha 16 was detected in the human progenitor B cell lines Reh and Nalm-6. Using the mu+, k-cell line BLIN-1 (pre-B cell phenotype) and its derived subclone 1E8 (surface mu+, k+; B-cell phenotype) G alpha 16 expression was found to disappear on transition from pre-B to B-cell differentiation stage. The analysis of a broad panel of human neoplastic B lymphocytes ranging from progenitor B-acute lymphatic leukemia (pre-pre-B-ALL), common acute leukemias (cALL), pre-B-ALL, mature B-ALL to low grade B-cell lymphoma (chronic lymphocytic leukemia of B-cell type, leukemic centrocytic non-Hodgkins lymphoma [NHL], hairy cell leukemia) showed that G alpha 16 expression is limited to progenitor and pre-B-ALL cells. Therefore, we conclude that within B-cell differentiation, G alpha 16 is expressed solely during early B cell ontogeny and downregulated during differentiation. Thus, G alpha 16 might be an important regulator involved in signaling processes in progenitor B cells.

scholarly journals Characterization of the DNA Methylome during Human B-Cell Differentiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4346-4346
Author(s):  
Marta Kulis ◽  
Simon Heath ◽  
Giancarlo Castellano ◽  
Renée Beekman ◽  
Angelika Merkel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Maturation ◽  
B Cell Differentiation ◽  
Dna Methylome ◽  
B Cell Maturation ◽  
Cell Subpopulations ◽  
Human B Cell

Abstract Introduction: Modulation of the DNA methylation landscape during cell differentiation is a well-established phenomenon. The B-cell lineage represents a paradigmatic cellular model to study the dynamic epigenome during cell development and specification because major B-cell maturation stages are well defined and display differential phenotypic and gene expression features. Furthermore, different B-cell subpopulations show different proliferation abilities, microenvironmental influences and life spans, providing a window of opportunity to study the epigenome in the context of multiple processes. Methods: We performed whole-genome bisulfite sequencing (WGBS), high-density methylation microarrays and gene expression profiling of ten purified human B-cell subpopulations spanning the entire differentiation program, ranging from uncommitted progenitors to terminally-differentiated plasma cells. Results: The results of both WGBS and methylation microarrays indicate that B-cell ontogenesis involves an extensive and gradual reconfiguration of the DNA methylome. We uncovered that non-CpG methylation at CpApC trinucleotides is present in progenitor cells and disappears upon B-cell commitment independently of CpG demethylation. CpG methylation, in contrast, changed extensively during the entire B-cell maturation program, with one quarter of all measured CpGs showing dynamic methylation. B-cell enhancers suffered more extensive methylation changes than promoter regions, especially in the early differentiation steps up to the germinal center B-cell (gcBC) stage, and their demethylation seemed to be mediated by binding of lineage-specific transcription factors. Enhancers with dynamic methylation were related to genes involved in a large B-cell network that showed high gene expression variability throughout differentiation. In highly proliferative gcBCs, we observed a shift of dynamic methylation from regulatory towards non-functional elements; gcBCs start to undergo global demethylation of late-replicating heterochromatic regions and methylation of polycomb-repressed regions. This signature becomes particularly extensive in long-lived memory B cells and plasma cells, indicating that these changes start in highly proliferative cells and then accumulate in non-proliferative cells with extended lifespan. Conclusion: Our epigenomic analysis of the B-cell differentiation program extends our knowledge on how the DNA methylome is modulated during cell specification and maturation and offers a resource for researchers in the field, both at global and single gene levels. Disclosures No relevant conflicts of interest to declare.

The EUROclass trial: defining subgroups in common variable immunodeficiency

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Claudia Wehr ◽  
Teemu Kivioja ◽  
Christian Schmitt ◽  
Berne Ferry ◽  
Torsten Witte ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Common Variable Immunodeficiency ◽  
Multicenter Trial ◽  
Memory B Cells ◽  
Granulomatous Disease ◽  
B Cell Differentiation ◽  
Cell Subpopulations ◽  
Common Variable

The heterogeneity of common variable immunodeficiency (CVID) calls for a classification addressing pathogenic mechanisms as well as clinical relevance. This European multicenter trial was initiated to develop a consensus of 2 existing classification schemes based on flowcytometric B-cell phenotyping and the clinical course. The clinical evaluation of 303 patients with the established diagnosis of CVID demonstrated a significant coincidence of granulomatous disease, autoimmune cytopenia, and splenomegaly. Phenotyping of B-cell subpopulations confirmed a severe reduction of switched memory B cells in most of the patients that was associated with a higher risk for splenomegaly and granulomatous disease. An expansion of CD21low B cells marked patients with splenomegaly. Lymphadenopathy was significantly linked with transitional B-cell expansion. Based on these findings and pathogenic consideration of B-cell differentiation, we suggest an improved classification for CVID (EUROclass), separating patients with nearly absent B cells (less than 1%), severely reduced switched memory B cells (less than 2%), and expansion of transitional (more than 9%) or CD21low B cells (more than 10%). Whereas the first group contains all patients with severe defects of early B-cell differentiation, severely reduced switched memory B cells indicate a defective germinal center development as found in inducible constimulator (ICOS) or CD40L deficiency. The underlying defects of expanded transitional or CD21low B cells remain to be elucidated. This trial is re-gistered at http://www.uniklinik-freiburg.de/zks/live/uklregister/Oeffentlich.html as UKF000308.

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 ◽  
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.

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.

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