Human naive B cells cultured with EL-4 T cells mimic a germinal center-related B cell stage before generating plasma cells. Concordant changes in Bcl-2 protein and messenger RNA levels.
The T cell-dependent B cell response in vivo occurs in organized microenvironments. Alternative routes exist in that early plasma cells are generated in the T zone while others emerge later from the germinal center (GC) reaction. We investigated whether B cell stages resembling those defined in vivo/ex vivo might be induced in an in vitro system in which naive human B cells are activated by EL-4 T cells and cytokines. Adult peripheral blood- or cord blood-derived B cells were found to mimic an early activated stage (CD38(low), IgD+, increased CD5+) followed by a centroblastic GC-related stage (CD38(int), CD77+, CD95(Fas)+, Bcl-2 protein(low)) before differentiating into morphologically typical, CD38(high), Fas- plasma cells of an immature type (Bcl-2(low), VLA-5-). The GC-related cells and the plasma cells exhibited spontaneous apoptosis in medium, the former also undergoing anti-Fas antibody-induced apoptosis in medium as well as during CD40L exposure in the EL-4 cultures. These Bcl-2(low) cells maintained a high viability in contact with EL-4 cells. Thus, some, major B cell stages with typical functional features as described for cells in vivo/ex vivo are sequentially generated in this in vitro system and the kinetics of the changes can be analyzed in a synchronized cell population. With regard to previous apparently conflicting observations on the Bcl-2 mRNA level in GC B cells, we performed competitive reverse-transcription polymerase chain reaction. Concordant changes in Bcl-2 mRNA and protein levels were found, i.e. during Bcl-2 down-regulation in the GC-related B cells in ongoing EL-4 cultures or in medium, and during a more modest up-regulation upon contact with fresh EL-4 cells. Regulation of Bcl-2 protein, therefore, predominantly occurred at the mRNA steady-state level.
Grimaître
M
M
Department of Medicine, Hôpitaux Universitaires de Genève, Hôpital Cantonal, Geneva, Switzerland.
Werner-Favre
C
C
Kindler
V
V
Zubler
R H
RH
D000328:Adult; D017209:Apoptosis; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D002454:Cell Differentiation; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005312:Fetal Blood; D015870:Gene Expression; D018858:Germinal Center / Q000166:cytology / Q000276:immunology; D006410:Hematopoiesis; D006801:Humans; D010950:Plasma Cells / Q000166:cytology / Q000276:immunology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D001613:beta 2-Microglobulin / Q000235:genetics; D019014:fas Receptor / Q000032:analysis
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; D019014:fas Receptor; D004247:DNA