This research will test a model of B cell triggering in which early biochemical events that follow crosslinking of surface immunoglobulin antigen receptors are on parallel pathways that lead to common mitogenic signals. The model derives from preliminary observations in this laboratory that the elevation of intracellular free calcium in response to receptor crosslinking appears to be composed of two separately triggered and separately inhibitable responses. The first goal of this research will be a more detailed characterization of the two calcium signals with respect to requirements for triggering and inhibition, in order to establish firmly whether they are on independent signal pathways. The second goal is to measure the calcium responses in cells which have been triggered by antigen and compare them with the responses elicited by the anti-immunoglobulin which is used to mimic antigen in the model system. In order to overcome problems associated with the infrequency of antigen-binding cells, a magnetic cell sorting procedure will be used to enrich the antigen-binding fraction, and a fluorescence microscope system will be used to study responses in single cells. The mechanism by which antigen activates resting B lymphocytes remains an important gap in our fundamental understanding of the initiation of an immune response. The innovative techniques for separation of antigen responsive resting B cells developed in this laboratory with previous NSF support provide the basis for the proposed further studies on this important problem.
