Antigen driven B cell responses occur within secondary lymphoid tissues. Actively cycling B cells migrate into follicles where germinal center (GC) responses develop. Since cycling GC B cells are programmed to undergo apoptosis, they must receive viability and/or proliferative signals from the GC environment in order to survival. The present study utilizes an in vitro model system to explore the biology of cycling B cells. For this purpose, small resting B cells isolated from the spleens of mice are activated in bulk culture for 2 or 4 days with lipopolysaccharide, and the actively cycling B cells recovered on 3-step Percoll gradients. The first survival signal required to protect cycling B cells from continuing into apoptosis is delivered by occupancy of the BCR by antigen. This represents the first step in the process of affinity maturation, since only the best fit clones can compete or limiting antigen. In the first specific aim, experiments are outlined to test the hypothesis that the CD19/CD21 co-receptor plays an important role during delivery of this initial survival signal to cycling B cells. For this purpose, cycling HEL transgenic B cells will be restimulated with soluble or immobilized HEL or a HEL-C3d fusion protein. Since C3d is the ligand for CD21, this fusion protein effectively recruits the CD19/CD21 co-receptor to sites of occupied BCR. Our previous studies have shown that strength of signal through BCR expressed by cycling B cells determines the pattern of Bcl-2 family expression. Cycling B cells express the pro-apoptotic proteins Bax and Bcl-XS. Cross-linking BCR on cycling B cells with soluble F(ab')2 anti-mu induces the appearance of the anti-apoptitic protein Bcl-XL, while optimal cross- linking of BCR by immobilized anti-mu induces both Bcl-XL and Bcl-2 anti-apoptotic proteins. Experiments described in the second specific aim will examine the biological consequences of these different BCR- induced patterns of survival/death proteins. Our previous studies have also shown that strength of signal through BCR expressed by cycling B cells determines their ability to respond to Th cell-derived signals. Experiments in the third specific aim will test the hypothesis that the BCR-derived signals accomplish this, in part, by regulating the composition of the CD40 signaling complex. In sum, these experiments will expand our understanding of how external signals impact on the fate of cycling B cells.
