Stromal cell independent B cell development in vitro: generation and recovery of autoreactive clones.
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.
Holl
T Matt
TM
Department of Immunology, DUMC 3010, Duke University Medical Center, Durham, NC 27710, USA.
Haynes
Barton F
BF
Kelsoe
Garnett
G
D000818:Animals; D000917:Antibody Formation; D000954:Antigens, Surface / Q000378:metabolism; D001323:Autoantibodies / Q000097:blood; D015551:Autoimmunity / Q000235:genetics; D053264:B-Cell Activating Factor / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology*; D001854:Bone Marrow Cells / Q000276:immunology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003181:Complement C4 / Q000172:deficiency / Q000235:genetics; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015322:Gene Rearrangement, B-Lymphocyte; D005838:Genotype; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007072:Immunoglobulin D / Q000378:metabolism; D007132:Immunoglobulin Isotypes / Q000235:genetics / Q000378:metabolism*; D007075:Immunoglobulin M / Q000378:metabolism; D016130:Immunophenotyping; D015851:Interleukin-7 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010641:Phenotype; D054448:Precursor Cells, B-Lymphoid / Q000276:immunology*; D017154:Stromal Cells / Q000276:immunology*; D013997:Time Factors; D051193:Toll-Like Receptors / Q000819:agonists
D000954:Antigens, Surface; D001323:Autoantibodies; D053264:B-Cell Activating Factor; C016759:C4b protein, mouse; D003181:Complement C4; D018398:Homeodomain Proteins; D007072:Immunoglobulin D; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D015851:Interleukin-7; D051193:Toll-Like Receptors; C064658:RAG-1 protein