Subpopulations of colony-forming B lymphocytes exhibit distinct in vitro growth characteristics

Human peripheral blood cells enriched for B lymphocytes were stimulated to focal proliferation in semisolid cultures with lymphocyte-conditioned media, Protein A (Prot A), lipopolysaccharide (LPS) and 2-mercaptoethanol (2-ME). After 6-8 days of incubation, two morphologically distinct colony types were observed. Type I colonies were diffusely proliferating aggregates within the agar layer, whereas another subset of B-cell-colony-forming cells (CFU-BL) formed round compact type II colonies which appeared to leave the agar layer and continued to proliferate in the liquid overlayer of our culture system. They reached maximum proliferation 2 days earlier than type I colonies. Cells derived from both colony types were positively identified as B lymphocytes by monoclonal antibodies using immunoperoxidase staining. In addition to this distinct growth pattern, both colony types exhibited different proliferative responses which were dependent on the kind of conditioned media used and the mitogen concentration. In secondary cultures both type I and type II colony-derived cells showed recloning capacity. However, after replating, both colony types gave rise to round compact type II colonies. These results demonstrate that there exist at least two subpopulations of colony-forming B lymphocytes, possibly one more primitive than the other, which can be distinguished by in vitro growth characteristics.