title: Sudden appearance of anti-protein IgG1-forming cell precursors early during primary immunization.
abstract: The anti-keyhole limpet hemocyanin (KLH) B-cell repertoire of unimmunized adult mice was examined by culture of splenocytes (generally 100-3000) at limiting dilution. Cells were polyclonally stimulated with Escherichia coli lipopolysaccharide (LPS) and an interleukin-4-containing lymphokine mixture in the presence of 3T3 fibroblast filler cells. After 7 days of culture, supernatants were examined for their content of anti-KLH IgM and IgG1 antibody by an enzyme-linked immunosorbent assay (ELISA). Parallel cultures of smaller numbers (generally 1-15) of splenocytes were examined to determine the cloning efficiency of B cells in terms of total IgM and IgG1 production. Whereas one spleen cell in 370 produced clones secreting anti-KLH IgM, only 1% of these produced IgG1 that could bind to KLH, despite the fact that about half of the clones switched to IgG1 production with these stimuli. In mice immunized with KLH, this situation did not change until day 5, when there was a sudden, explosive emergence of B cells that could form clones secreting anti-KLH IgG1. The absolute number of such cells in the spleen was found to rise by a factor of 350 between days 3 and 7 of immunization. Moreover, the median amount of IgG1 antibody formed per clone and binding to KLH also rose markedly. In contrast, neither the numbers nor the median KLH-binding antibody content of anti-KLH IgM clones changed significantly after immunization. The results show that the repertoire of anti-protein B cells detected through IgM formation in ELISA consists chiefly of cells producing antibody of low avidity and of doubtful in vivo significance. Assuming that the small proportion of these cells making antibody that is of sufficient avidity to bind as the IgG1 isotype are the ancestors of the many such cells found on day 7 of the primary immune response, one would have to postulate a very high recruitment and/or division rate to account for the increase in numbers and avidity that occurs. It is possible that the anti-KLH IgG1 precursors that suddenly emerge are the results of early variable region gene (V) mutations in B cells. Moreover, it is not excluded that they represent products of a subset of B cells different from those that give rise to the primary in vitro anti-KLH IgM response. The findings have implications for theories of B-cell tolerance.
authors: Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
authors: G J
authors: GJ
authors: Nossal
authors: C
authors: C
authors: Riedel
mesh_terms: D000818:Animals; D000917:Antibody Formation; D001402:B-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007114:Immunization; D007074:Immunoglobulin G / Q000096:biosynthesis*; D007075:Immunoglobulin M / Q000096:biosynthesis; D015847:Interleukin-4; D007378:Interleukins / Q000276:immunology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D008808:Mice, Inbred CBA
chemical_list: D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007378:Interleukins; D008070:Lipopolysaccharides; D015847:Interleukin-4