V(D)J recombinatorial repertoire diversification during intraclonal pro-B to B-cell differentiation

Abstract The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rearrangements that occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was provided by the identification of a human acute lymphoblastic leukemic cell line that undergoes spontaneous differentiation from pro-B cell to the pre-B and B-cell stages with concomitant changes in the gene expression profile that normally occur during B-cell differentiation. After confirming the clonality of the progressively differentiating cells, an analysis of immunoglobulin genes and transcripts indicated that pro-B cell members marked by the same DJ rearrangement generated daughter B cells with multiple VH and VL gene segment rearrangements. These findings validate the principle of intraclonal V(D)J diversification during B-cell generation and define a manipulable model of human B-cell differentiation.

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Delineation of Human B Cell Differentiation: Immunological and Molecular Characterization of Human B Cell Differentiation Factor (BSF-2)

Mechanisms of Lymphocyte Activation and Immune Regulation - Advances in Experimental Medicine and Biology ◽

10.1007/978-1-4684-5323-2_17 ◽

1987 ◽

pp. 177-188 ◽

Cited By ~ 4

Author(s):

Tadamitsu Kishimoto ◽

Toshio Hirano ◽

Hitoshi Kikutani ◽

Atsushi Muraguchi

Keyword(s):

Cell Differentiation ◽

B Cell ◽

Molecular Characterization ◽

B Cell Differentiation ◽

Differentiation Factor ◽

Cell Differentiation Factor ◽

Human B Cell

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Induction of human B cell differentiation by Fc region activators

Clinical Immunology and Immunopathology ◽

10.1016/0090-1229(89)90133-5 ◽

1989 ◽

Vol 50 (2) ◽

pp. 251-263 ◽

Cited By ~ 4

Author(s):

Monte V. Hobbs ◽

Richard A. Houghten ◽

Jodee A. Janda ◽

William O. Weigle ◽

Edward L. Morgan

Keyword(s):

Cell Differentiation ◽

B Cell ◽

B Cell Differentiation ◽

Human B Cell

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Molecular structure and immunological function of human B cell differentiation factor (BSF2)

Molecular Basis of Lymphokine Action ◽

10.1007/978-1-4612-4598-8_12 ◽

1987 ◽

pp. 123-136 ◽

Cited By ~ 1

Author(s):

Tadamitsu Kishimoto ◽

Tetsuya Taga ◽

Kiyoshi Yasukawa ◽

Yasuo Watanabe ◽

Tadashi Matsuda ◽

...

Keyword(s):

Molecular Structure ◽

Cell Differentiation ◽

B Cell ◽

B Cell Differentiation ◽

Immunological Function ◽

Differentiation Factor ◽

Cell Differentiation Factor ◽

Human B Cell

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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.

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