Activation of human B lymphocytes by nanogram concentrations of anti-IgM-dextran conjugates

Surface immunoglobulin (sIg) cross-linking on B lymphocytes by high concentrations of anti-Ig antibody has been used to mimic antigen-stimulated B cell activation. In order to develop a system to study sIg-mediated T cell-independent B cell activation using low concentrations of anti-Ig antibody that more closely resemble the concentrations of antigen that are achieved under in vivo conditions, we conjugated monoclonal anti-human IgM antibody (anti-mu) to dextran (molecular weight 2 X 10(6)) thereby increasing its valency. This dextran conjugate (anti-mu-Dex) stimulated comparable levels of thymidine incorporation and B cell size increases as were seen with unconjugated anti-mu but at 100- to 1000-fold lower concentrations. Anti-mu-Dex also stimulated increases in intracellular ionized calcium ([Ca2+]i) in a higher percentage of cells, of greater magnitude and of longer duration than that stimulated by unconjugated anti-mu. Interestingly, there was no direct correlation between the increases in [Ca2+]i that were stimulated by anti-mu-Dex and its ability to stimulate B cell proliferation. The concentrations of anti-mu-Dex (10 micrograms/ml) that led to the highest increase in [Ca2+]i resulted in thymidine incorporation that was no greater than that of medium control, whereas 0.01 to 0.1 microgram/ml stimulated significant thymidine incorporation with 50% lower levels of stimulation of [Ca2+]i. These data demonstrate that anti-mu-Dex is a potent activator of human B lymphocytes, is effective even at ng/ml concentrations which over a 2-h time period do not induce detectable modulation of sIg, and its stimulation of B cells into G1 and S may not be directly related to its ability to stimulate increases in levels of [Ca2+]i.