Stromal cell independent B cell development in vitro: generation and recovery of autoreactive clones

J Immunol Methods. 2010 Mar 31;354(1-2):53-67. doi: 10.1016/j.jim.2010.01.007. Epub 2010 Jan 28.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibody Formation
  • Antigens, Surface / metabolism
  • Autoantibodies / blood
  • Autoimmunity* / genetics
  • B-Cell Activating Factor / metabolism
  • B-Lymphocytes / immunology*
  • Bone Marrow Cells / immunology*
  • Cell Culture Techniques
  • Cell Differentiation* / genetics
  • Cell Proliferation*
  • Cell Survival
  • Cells, Cultured
  • Complement C4 / deficiency
  • Complement C4 / genetics
  • Flow Cytometry
  • Gene Expression Profiling
  • Gene Rearrangement, B-Lymphocyte
  • Genotype
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Immunoglobulin D / metabolism
  • Immunoglobulin Isotypes / genetics
  • Immunoglobulin Isotypes / metabolism*
  • Immunoglobulin M / metabolism
  • Immunophenotyping
  • Interleukin-7 / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mice, Transgenic
  • Phenotype
  • Precursor Cells, B-Lymphoid / immunology*
  • Stromal Cells / immunology*
  • Time Factors
  • Toll-Like Receptors / agonists

Substances

  • Antigens, Surface
  • Autoantibodies
  • B-Cell Activating Factor
  • C4b protein, mouse
  • Complement C4
  • Homeodomain Proteins
  • Immunoglobulin D
  • Immunoglobulin Isotypes
  • Immunoglobulin M
  • Interleukin-7
  • Toll-Like Receptors
  • RAG-1 protein