Activating B cell signaling with defined multivalent ligands

Depending on the stimuli they encounter, B lymphocytes engage in signaling events that lead to immunity or tolerance. Both responses are mediated through antigen interactions with the B cell antigen receptor (BCR). Antigen valency is thought to be an important parameter in B cell signaling, but systematic studies are lacking. To explore this issue, we synthesized multivalent ligands of defined valencies using the ring-opening metathesis polymerization (ROMP). When mice are injected with multivalent antigens generated by ROMP, only those of high valencies elicit antibody production. These results indicate that ligands synthesized by ROMP can activate immune responses in vivo. All of the multivalent antigens tested activate signaling through the BCR. The ability of antigens to cluster the BCR, promote its localization to membrane microdomains, and augment intracellular Ca2+ concentration increases as a function of antigen valency. In contrast, no differences in BCR internalization were detected. Our results indicate that differences in the antigenicity of BCR ligands are related to their ability to elicit increases in intracellular Ca2+ concentration. Finally, we observed that unligated BCRs cluster with BCRs engaged by multivalent ligands, a result that suggests that signals mediated by the BCR are amplified through receptor arrays. Our data suggest a link between the mechanisms underlying signal initiation by receptors that must respond with high sensitivity.