title: Maintenance of human germinal center B cells in vitro.
abstract: The ability to maintain germinal center (GC) B cells in culture should facilitate studies on the molecular and cellular events which accompany affinity maturation and the generation of memory in T-dependent responses. We have investigated the ability of cytokines to maintain human tonsillar GC B cells (IgD-/CD39-/CD38+/CD77+) in the "CD40 culture system". In the absence of added cytokines, CD40 monoclonal antibody held on CD32-transfected L cells effectively sustained DNA synthesis in GC B cells for a maximum 3 to 4 days. Of the following cytokines (interleukin-1 beta [IL-1 beta], IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, and stem cell factor), only IL-2 and IL-4 provided a significant enhancement to DNA synthesis in the CD40 culture system; this was modest and short-term. Following a study on the cooperative activity between pairs of cytokines, triple combinations were identified that could maintain high levels of GC B-cell stimulation for at least 10 days. IL-10 was a common component of these synergistic cytokine cocktails, which were IL-10 + IL-4 + IL-7; IL-10 + IL-3 + IL-7; IL-10 + IL-1 beta + IL-2; IL-10 + IL-1 beta + IL-3, and IL-10 + IL-3 + IL-6. Culture of GC B cells with these cytokine combinations resulted in a net increase in viable cell numbers of 50% to 100% whereas total cell numbers increased up to fourfold. Cells recovered from these cultures retained a GC B-cell phenotype with a significant proportion being CD38+/CD44-, features characteristic of centroblasts. Studies with metabolically inactive CD32-L cells supported a role for stromal cell-derived soluble factors in maintaining GC B cells in vitro.
authors.affiliation: Department of Immunology, Medical School, University of Birmingham, UK.
authors.forename: J D
authors.initials: JD
authors.lastname: Pound
authors.forename: J
authors.initials: J
authors.lastname: Gordon
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001402:B-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D019013:CD40 Antigens / Q000276:immunology / Q000502:physiology; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects / Q000276:immunology; D004338:Drug Combinations; D004357:Drug Synergism; D018858:Germinal Center / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology; D007739:L Cells / Q000378:metabolism; D051379:Mice; D017452:Receptors, IgG / Q000235:genetics / Q000502:physiology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D016207:Cytokines; D004338:Drug Combinations; D017452:Receptors, IgG; D016753:Interleukin-10