Proliferation and differentiation of single hapten-specific B lymphocytes is promoted by T-cell factor(s) distinct from T-cell growth factor

Abstract

Hapten-specific B lymphocytes reactive to fluorescein were prepared from mouse spleen, placed singly in 10-microliters culture wells, and stimulated with fluorescein-polymerized flagellin in the presence of conditioned media (CM) from various concanavalin A-stimulated cloned T-cell tumors or hybridomas. Antigen plus appropriate CM triggered 5-9% of the B cells into both clonal proliferation and differentiation into antibody-forming cells. Antigen alone stimulated 0.5-0.8% of B cells and CM alone stimulated less than 0.1%. This bioactivity was termed B-cell growth and differentiation factor(s) (BGDF). Four CM rich in T-cell growth factor (TCGF)--namely, CM from spleen and the lines EL4, T6, and 123--contained BGDF. The lines T19.1 and WEHI-3 lacked BGDF and TCGF. Four lines of evidence suggested that BGDF and TCGF were distinct molecules. First, the BGDF/TCGF ratios in the various CM varied. Second, on gel filtration, TCGF eluted as a sharp peak corresponding to a Mr of about 35,000, whereas BGDF eluted over a range corresponding to a Mr of 25,000-60,000. Third, the activity of TCGF in EL4-CM was markedly reduced by treatment with guanidine HCl while BGDF activity was not. Fourth, BGDF showed more heterogeneity than TCGF on hydrophobic chromatography. All CM or fractions active in promoting B-cell division also promoted differentiation to antibody-forming cells. These results provide unequivocal evidence that antigen and a T-cell product can synergize to directly activate a single B lymphocyte.