Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

Marsman, Casper; Jorritsma, Tineke; Brinke, Anja ten; Ham, S. Marieke van

doi:10.3390/cells9122633

Cited by 23 publications

(18 citation statements)

References 57 publications

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“…To address further the link between the extracellular signals provided and the induction of a transcriptional program that efficiently induces ASC differentiation, we investigated the kinetics of phosphorylation of STAT3 (pSTAT3; Figure 6 A), the signaling molecule positively regulating PRDM1 expression and consequently negatively regulating the B-cell transcriptional program ( Figure 5 A). In line with previous data [ 33 ], the presence of IL-21 induced a strong expression of pSTAT3. At the start of culture, pSTAT3 levels in the presence of IL-21 steadily increased and reached maximum levels after 3 days, before return to baseline 3 days later ( Figure 6 B,C).…”

Section: Resultssupporting

confidence: 93%

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells

Unger

Verstegen

Marsman

et al. 2021

Cells

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High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

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Section: Resultssupporting

confidence: 93%

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells

Unger

Verstegen

Marsman

et al. 2021

Cells

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“…Staining procedures were performed as previously described ( 59 ). In short, harvested and pooled cultures were kept on melting ice at all times.…”

Section: Resultsmentioning

confidence: 99%

Oxygen level is a critical regulator of human B cell differentiation and IgG class switch recombination

et al. 2022

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The generation of high-affinity antibodies requires an efficient germinal center (GC) response. As differentiating B cells cycle between GC dark and light zones they encounter different oxygen pressures (pO2). However, it is essentially unknown if and how variations in pO2 affect B cell differentiation, in particular for humans. Using optimized in vitro cultures together with in-depth assessment of B cell phenotype and signaling pathways, we show that oxygen is a critical regulator of human naive B cell differentiation and class switch recombination. Normoxia promotes differentiation into functional antibody secreting cells, while a population of CD27++ B cells was uniquely generated under hypoxia. Moreover, time-dependent transitions between hypoxic and normoxic pO2 during culture - reminiscent of in vivo GC cyclic re-entry - steer different human B cell differentiation trajectories and IgG class switch recombination. Taken together, we identified multiple mechanisms trough which oxygen pressure governs human B cell differentiation.

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“…Future research is needed to define these autonomous factors and address signaling pathways involved in both beneficial and unwanted plasma cell development. Comparing patients and healthy donors in optimized cultures and assays that detect gene expression and post-translational modifications such as phosphorylation or ubiquitination by intracellular staining methods ( 43 ) may aid in these research questions. The TD and TI assays described here in condition II (and II.2), being 2,500 CD19 + B cells stimulated with CD40L and IL-21, and in condition IV (and IV.2), being 25,000 CD19 + B cells stimulated with CpG, IL-2, and possibly BAFF, support the efficient differentiation of human primary B cells into plasma cells, with warranted B-cell expansion, proliferation, and quantifiable production of IgG, IgA, and IgM.…”

Section: Discussionmentioning

confidence: 99%

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

Marsman

Verhoeven²,

Koers³

et al. 2022

Front. Immunol.

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Background/MethodsFor 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 make the 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 peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were 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.ResultsThis study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells 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 2,500 and 25,000 B–cells per culture well for the 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 a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The 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 were accomplished when starting from fresh or cryopreserved samples.ConclusionOur methodology demonstrates optimized TD and TI stimulation protocols for more in-depth 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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Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

Cited by 23 publications

References 57 publications

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells

Oxygen level is a critical regulator of human B cell differentiation and IgG class switch recombination

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

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