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

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Abstract
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 Tcell‐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‐μ) to dextran (molecular weight 2 × 106) thereby increasing its valency. This dextran conjugate (anti‐μ‐Dex) stimulated comparable levels of thymidine incorporation and B cell size increases as were seen with unconjugated anti‐μ but at 100‐ to 1000‐fold lower concentrations. Anti‐μ‐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‐μ. Interestingly, there was no direct correlation between the increases in [Ca2+]i that were stimulated by anti‐μ‐Dex and its ability to stimulate B cell proliferation. The concentrations of anti‐μ‐Dex (10 μg/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 μg/ml stimulated significant thymidine incorporation with 50% lower levels of stimulation of [Ca2+]i. These data demonstrate that anti‐μ‐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.