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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Banchereau, J., de Paoli, P., Valle, A., Garcia, E. & Rousset, F. Long-term human B cell lines dependent on interleukin-4 and antibody to CD40. Science 251, 70–72 (1991).
Kim, C.H. et al. Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. J. Exp. Med. 193, 1373–1381 (2001).
Good, K.L., Bryant, V.L. & Tangye, S.G. Kinetics of human B cell behavior and amplification of proliferative responses following stimulation with IL-21. J. Immunol. 177, 5236–5247 (2006).
Ettinger, R. et al. IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. J. Immunol. 175, 7867–7879 (2005).
Kuo, T.C. et al. Repression of Bcl-6 is required for the formation of human memory B cells in vitro. J. Exp. Med. 204, 819–830 (2007).
Shapiro-Shelef, M. et al. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19, 607–620 (2003).
Dent, A.L., Shaffer, A.L., Yu, X., Allman, D. & Staudt, L.M. Control of inflammation, cytokine expression, and germinal center formation by Bcl-6. Science 276, 589–592 (1997).
Fukuda, T. et al. Disruption of the Bcl6 gene results in an impaired germinal center formation. J. Exp. Med. 186, 439–448 (1997).
Phan, R.T. & Dalla-Favera, R. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature 432, 635–639 (2004).
Shapiro-Shelef, M. & Calame, K. Regulation of plasma-cell development. Nat. Rev. Immunol. 5, 230–242 (2005).
Shvarts, A. et al. A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative p19(ARF)-p53 signaling. Genes Dev. 16, 681–686 (2002).
Scheeren, F.A. et al. STAT5 regulates the self-renewal capacity and differentiation of human memory B cells and controls Bcl-6 expression. Nat. Immunol. 6, 303–313 (2005).
Diehl, S.A. et al. STAT3-mediated up-regulation of BLIMP1 is coordinated with BCL6 down-regulation to control human plasma cell differentiation. J. Immunol. 180, 4805–4815 (2008).
Liu, Y.J. & Arpin, C. Germinal center development. Immunol. Rev. 156, 111–126 (1997).
Niiro, H. & Clark, E.A. Regulation of B cell fate by antigen-receptor signals. Nat. Rev. Immunol. 2, 945–956 (2002).
Bryant, V.L. et al. Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells. J. Immunol. 179, 8180–8190 (2007).
Hall, C.B. et al. The burden of respiratory syncytial virus infection in young children. N. Engl. J. Med. 360, 588–598 (2009).
Johnson, S. et al. A direct comparison of the activities of two humanized respiratory syncytial virus monoclonal antibodies: MEDI-493 and RSHZl9. J. Infect. Dis. 180, 35–40 (1999).
Sidwell, R.W. & Barnard, D.L. Respiratory syncytial virus infections: recent prospects for control. Antiviral Res. 71, 379–390 (2006).
Domachowske, J.B., Bonville, C.A. & Rosenberg, H.F. Animal models for studying respiratory syncytial virus infection and its long term effects on lung function. Pediatr. Infect. Dis. J. 23, S228–S234 (2004).
Peled, J.U. et al. The biochemistry of somatic hypermutation. Annu. Rev. Immunol. 26, 481–511 (2008).
Sayegh, C.E., Quong, M.W., Agata, Y. & Murre, C. E-proteins directly regulate expression of activation-induced deaminase in mature B cells. Nat. Immunol. 4, 586–593 (2003).
Dong, C., Temann, U.A. & Flavell, R.A. Cutting edge: critical role of inducible costimulator in germinal center reactions. J. Immunol. 166, 3659–3662 (2001).
Shinkura, R. et al. Separate domains of AID are required for somatic hypermutation and class-switch recombination. Nat. Immunol. 5, 707–712 (2004).
McBride, K.M. et al. Regulation of class switch recombination and somatic mutation by AID phosphorylation. J. Exp. Med. 205, 2585–2594 (2008).
Traggiai, E. et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 10, 871–875 (2004).
Dykstra, M.L., Longnecker, R. & Pierce, S.K. Epstein-Barr virus coopts lipid rafts to block the signaling and antigen transport functions of the BCR. Immunity 14, 57–67 (2001).
Wrammert, J. et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667–671 (2008).
Zwick, M.B., Gach, J.S. & Burton, D.R. A welcome burst of human antibodies. Nat. Biotechnol. 26, 886–887 (2008).
Deinhardt, F., Falk, L.A. & Wolfe, L.G. Transformation of nonhuman primate lymphocytes by Epstein-Barr virus. Cancer Res. 34, 1241–1244 (1974).
Jaleco, A.C. et al. Genetic modification of human B cell development: B cell development is inhibited by the dominant negative helix loop helix factor Id3. Blood 94, 2637–2646 (1999).
Heemskerk, M.H. et al. Enrichment of an antigen-specific T cell response by retrovirally transduced human dendritic cells. Cell. Immunol. 195, 10–17 (1999).
Smit, L.A. et al. Expression of activation-induced cytidine deaminase is confined to B cell non-Hodgkin′s lymphomas of germinal-center phenotype. Cancer Res. 63, 3894–3898 (2003).
Prince, G.A., Prieels, J.P., Slaoui, M. & Porter, D.D. Pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (Sigmodon hispidus). Lab. Invest. 79, 1385–1392 (1999).
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.
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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