Preprint Article Version 2 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 18 November 2021 / Approved: 19 November 2021 / Online: 19 November 2021 (14:50:39 CET)
Version 2 : Received: 19 November 2021 / Approved: 22 November 2021 / Online: 22 November 2021 (14:10:02 CET)

How to cite: Marsman, C.; Verhoeven, D.; Koers, J.; Rispens, T.; ten Brinke, A.; van Ham, S.M.; W. Kuijpers, T. Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells . Preprints 2021, 2021110365 (doi: 10.20944/preprints202111.0365.v2). Marsman, C.; Verhoeven, D.; Koers, J.; Rispens, T.; ten Brinke, A.; van Ham, S.M.; W. Kuijpers, T. Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells . Preprints 2021, 2021110365 (doi: 10.20944/preprints202111.0365.v2).

Abstract

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.

Keywords

B cell activation; B cell differentiation; plasma cells; CD40L; IL-21; CpG; IL-2

Subject

LIFE SCIENCES, Immunology

Comments (1)

Comment 1
Received: 22 November 2021
Commenter: Casper Marsman
Commenter's Conflict of Interests: Author
Comment: No changes. Just added the other co-authors.
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