Abstract
We describe and characterize a stromal cell independent culture system that efficiently supports pro-B cell to IgM+ B cell development with near normal levels of IgH and Igkappa diversity. Pro-B cells present in non-adherent bone marrow cells proliferate in the presence of IL-7 and subsequent to the removal of IL-7 and addition of BAFF, differentiate normally into IgM+ B cells. B cell development in vitro closely follows the patterns of development in vivo with culture-derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. IgM+ CD B cells respond to TLR stimulation by proliferation and differentiation into antibody-secreting cells. Self-reactive IgM+ B cell development is blocked in 3H9 IgH knockin mice; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of "forbidden", autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4(-/-) animals following the reconstitution of RAG1(-/-) mice with IgM+ CD cells derived from BL/6 mice.
2010 Elsevier B.V. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antibody Formation
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Antigens, Surface / metabolism
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Autoantibodies / blood
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Autoimmunity* / genetics
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B-Cell Activating Factor / metabolism
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B-Lymphocytes / immunology*
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Bone Marrow Cells / immunology*
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Cell Culture Techniques
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Cell Differentiation* / genetics
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Cell Proliferation*
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Cell Survival
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Cells, Cultured
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Complement C4 / deficiency
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Complement C4 / genetics
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Flow Cytometry
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Gene Expression Profiling
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Gene Rearrangement, B-Lymphocyte
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Genotype
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Homeodomain Proteins / genetics
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Homeodomain Proteins / metabolism
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Immunoglobulin D / metabolism
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Immunoglobulin Isotypes / genetics
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Immunoglobulin Isotypes / metabolism*
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Immunoglobulin M / metabolism
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Immunophenotyping
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Interleukin-7 / metabolism
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Mice
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Mice, Inbred C57BL
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Mice, Knockout
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Mice, Transgenic
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Phenotype
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Precursor Cells, B-Lymphoid / immunology*
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Stromal Cells / immunology*
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Time Factors
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Toll-Like Receptors / agonists
Substances
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Antigens, Surface
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Autoantibodies
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B-Cell Activating Factor
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C4b protein, mouse
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Complement C4
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Homeodomain Proteins
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Immunoglobulin D
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Immunoglobulin Isotypes
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Immunoglobulin M
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Interleukin-7
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Toll-Like Receptors
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RAG-1 protein