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

Abstract

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 white others emerge later from the germinal center (GC) reaction. We investigated whether B cell stages resembling those defined in vivolex 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 vivolex 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.