J Immunol Methods. 2010 Mar 31; 354(43467):53-67.

PubMed ID: 20109461

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

Holl TM, Haynes BF, Kelsoe G

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.