title: An in vitro approach for the characterization of the cycling B cell response.
abstract: Because isolation of sufficient numbers of cycling, germinal center B cells from mice for biochemical characterization of BCR-derived signals can be problematic, we have designed an experimental approach for generating large numbers of cycling B cells for further study. In the experiments reported here, small, resting B cells were polyclonally stimulated with lipopolysaccharide (LPS), and cycling B cells isolated as two bands on three-step Percoll gradients. Cycling B cells isolated at Days 2, 4, or 6 of preactivation showed an increased expression of Fas receptor and peanut agglutinin binding, with a concomitant decrease in sIgD positivity. These cells phenotypically resembled extrafollicular or early germinal center B cells. These cycling B cells were used to study the functional consequences of differential signaling through the BCR. Strong cross-linking of BCR, by restimulation of cycling normal B cells with either immobilized or soluble F(ab')2 anti-mu and cycling hen egg lysozyme (HEL) transgenic B cells with either soluble or immobilized HEL, extended cellular proliferation by 2-3 d. In contrast, cycling B cells either restimulated with soluble, whole anti-mu (to mimic binding of soluble immune complexes) or cultured in the absence of restimulation (to mimic cycling B cells not competitive for antigen) resulted in the rapid exit of the cells from cycle. This system will enable the molecular and biochemical characterization of signal delivery to cycling B cells.
authors.affiliation: Department of Microbiology and Immunology, University of Kentucky Medical Center, Lexington 40536-0084, USA.
authors.forename: B T
authors.initials: BT
authors.lastname: Pittner
authors.forename: M W
authors.initials: MW
authors.lastname: Mullins
authors.forename: S
authors.initials: S
authors.lastname: Reid
authors.forename: E C
authors.initials: EC
authors.lastname: Snow
mesh_terms: D000818:Animals; D016175:B-Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology; D002453:Cell Cycle; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry; D016130:Immunophenotyping; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic; D013154:Spleen / Q000166:cytology
chemical_list: D008070:Lipopolysaccharides