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Generation of stable monoclonal antibody–producing B cell receptor–positive human memory B cells by genetic programming

A Corrigendum to this article was published on 06 December 2016

This article has been updated

Abstract

The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)–positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.

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Figure 1: Overexpression of Bcl-6 and Bcl-xL confers a high proliferative capacity and fixed differentiation phenotype to peripheral blood CD27+IgG+ memory B cells.
Figure 2: CD27+ memory peripheral blood cells acquire a stable germinal center–like phenotype after transduction with Bcl-6 and Bcl-xL and subsequent culturing.
Figure 3: Generation and characterization of tetanus toxin (TT)-specific monoclonal human B cell lines.
Figure 4: Isolation of high-affinity, broadly RSV-neutralizing antibodies from RSV-specific B cell clones.
Figure 5: Expression and activity of AID in Bcl-6– and Bcl-xL–transduced cells.

Change history

  • 18 August 2016

    In the version of this article initially published, the article did not mention some restrictions on the availability of reagents. Please note that the retroviral vectors containing BCL-6 and BCL-xL have been generated by a for-profit company, AIMM Therapeutics, which makes the plasmids available. Obtaining the plasmids requires an MTA (http://www.aimmtherapeutics.com/partnering/academic-collaboration/) that includes financial obligations.

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Acknowledgements

We thank B. Hooijbrink for his excellent help with FACS sorting and maintenance of the flow cytometry facility, J. Boes for her excellent work on virus titrations at the Netherlands Vaccine Institute and R. Molenkamp and D. Pajkrt of the Department of Clinical Virology of the Academic Medical Center for helpful discussions. Human cDNA encoding Bcl-xL was kindly provided by S. Korsmeyer (Howard Hughes Medical Institute, Harvard Medical School). Id-3 was a gift from C. Murre (University of California, San Diego). CD40L-L cells were from J. Banchereau (Baylor University). S.A.D. is supported by US National Institutes of Health–National Institute of Allergy and Infectious Diseases grant F32AI063846. M.V.L. is supported by grant OZF-02-004 from the Wilhelmina Research Fund.

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M.J.K., S.A.D. and T.B. performed experiments, analyzed data and wrote the paper. H.S. and T.B. organized the research and wrote the paper. E.Y., A.Q.B. and C.M.M.v.G. performed experiments. M.V.L., G.M.v.B., H.M., A.R. and W.M.J.M.B. contributed valuable reagents and developed assays. M.N.W. performed cotton rat experiments. F.A.S. analyzed data and provided valuable suggestions.

Corresponding author

Correspondence to Tim Beaumont.

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Competing interests

S.A.D., F.A.S., E.Y., M.J.K., H.S. and T.B. are listed on patent applications (international patent PCT/NL2008/050333 entitled 'RSV specific binding molecules and means for producing them' and PCT/NL2005/000848 'Means and methods for influencing the stability of cells' (to the patent offices of Japan, the US, Brazil, Canada, Europe, Australia and New Zealand) describing the RSV-specific antibodies and describing the B cell immortalization technology using Bcl-6 and Bcl-xL technology. M.J.K., E.Y., A.Q.B., C.M.M.v.G., T.B. and H.S. are employees of AIMM Therapeutics. H.S. has personal financial interests in AIMM Therapeutics.

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Kwakkenbos, M., Diehl, S., Yasuda, E. et al. Generation of stable monoclonal antibody–producing B cell receptor–positive human memory B cells by genetic programming. Nat Med 16, 123–128 (2010). https://doi.org/10.1038/nm.2071

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