AN IN-VITRO APPROACH FOR THE CHARACTERIZATION OF THE CYCLING B-CELL RESPONSE

Because isolation of sufficient numbers of cycling, germinal center B cells from mice for biochemical characterization of BCR-derived signal s can be problematic, we have designed an experimental approach for ge nerating large numbers of cycling B cells for further study. In the ex periments reported here. small, resting B cells were polyclonally stim ulated with lipopolysaccharide (LPS), and cycling B cells isolated as two bands on three-step Percoll gradients. Cycling B cells isolated at Days 2, 4, or 6 of preactivation showed an increased expression of Fa s receptor and peanut agglutinin binding, with a concomitant decrease in sIgD positivity. These cells phenotypically resembled extrafollicul ar or early germinal center B cells. These cycling B cells were used t o study the functional consequences of differential signaling through the BCR. Strong cross-linking of BCR, by restimulation of cycling norm al B cells with either immobilized or soluble F(ab')(2) anti-mu and cy cling hen egg lysozyme (HEL) transgenic B cells with either soluble or immobilized HEL, extended cellular proliferation by 2-3 d. In contras t, cycling B cells either restimulated with soluble, whole anti-mu (to mimic binding of soluble immune complexes) or cultured in the absence of restimulation (to mimic cycling B cells not competitive for antige n) resulted in the rapid exit of the cells from cycle. This system wil l enable the molecular and biochemical characterization of signal deli very to cycling B cells.