Label-free optical biosensor for on-line monitoring the integrated response of human B cells upon the engagement of stimulatory and inhibitory immune receptors

In this study a novel, label-free method using the evanescent field based resonance waveguide grating technology was applied to examine B cell activation. We successfully immobilized non-adherent B cells on the surface of the biosensors, without the ligation of any specific receptors or adhesion molecules. This way we were able to demonstrate that engagement of the antigen specific B cell receptors (BCR) induced reproducible dynamic mass redistribution (DMR) inside the cells as a measure of receptor activation. The initiated DMR response proved to be specific, since only antibodies recognizing the BCR could generate the response; neither the assay-buffer, nor high concentration of indifferent proteins or non-specific antibodies had any effect. The measure of cell activation was sensitive, concentration dependent, and specifically and dose-dependently inhibited by the Syk inhibitor BAY 61-3606. The BCR-triggered DMR response was evoked from three human Epstein-Barr virus (EBV) negative B cell lines, but could not be elicited in two EBV-positive BL cell lines, where the presence of the EBV-derived LMP2A protein desensitizes the cells' response to the BCR-induced signaling. Parallel engagement of the inhibitory FcγRIIB together with the BCR resulted in the inhibition of the activation of B cells demonstrating that the DMR response mirrors the expected interaction between the downstream signaling events of the two receptors. As a multi-target profiling procedure, this label-free RWG technology is applicable for the study of complex cellular signaling via the analysis of integrated real time signature of DMR. Therefore, our work opens new avenues to study complex signaling events and to decipher interactions within the signaling network during B cell activation.