SummaryThe mature, resting immunoglobulin (Ig) M, IgD + B lymphocyte can be induced by T cells to proliferate, switch isotype, and differentiate into Ig-secreting or memory cells. Furthermore, B cell activation results in the de novo expression or loss of a number of cell surface molecules that function in cell recirculation or further interaction with T cells. Here, a novel fluorescent technique reveals that T-dependent B cell activation induces cell surface changes that correlate with division cycle number. Furthermore, striking stepwise changes are often centered on a single round of cell division. Particularly marked was the consistent increase in IgG1 + B cells after the second division cycle, from an initial level of <3% IgG1 + to a plateau of~40% after six cell divisions. The relationship between the percentage oflgG1 + B cells and division number was independent of time after stimulation, indicating a requirement for cell division in isotype switching. IgD expression became negative after four divisions, and a number of changes centered on the sixth division, including the loss of IgM, CD23, and B220. The techniques used here should prove useful for tracking other differentiation pathways and for future analysis of the molecular events associated with stepwise differentiation at the single cell level.activation by T cells, mature B lymphocytes prorate and develop into Ig-secreting or memory cells (1). T cell-activated B cells also alter their functional characteristics such as Ig isotype, and expression of cell surface markers in response to cell contact and cytokine-mediated signals. The best studied example is the response to the cytokine IL-4, which induces the activated B cell to switch isotype from IgM to IgG1 and IgE (2, 3). As B cell differentiation is usually associated with mitosis, it has been difficult to directly assess whether cell division is required for differentiation, although a number of previous reports have linked the isotype switching mechanism with cell division (4-8). A technique for simultaneously tracking the division cycle history of stimulated cells and examining the cell surface phenotype has been developed by Lyons and Parish (9). Here, we use this method to track the relationship between division cycle number and B cell Ig isotype expression induced by the combination of plasma membranes from activated T cells and T cell-derived lymphokines (10). The results indicate that expression of surface IgG1 and other markers of B cell activation correlate with cell division number, making possible further molecular analysis of the process. Materials and MethodsPreparation and Stimulation of B Cells. Small, dense resting B cells were prepared by Percoll density gradient ~om anti-Thyl, -CD4, -CD8, and complement-treated CBA/H mouse spleens as described (10). B cells to be labeled with carboxyfluorescein, diacerate succinimidyl ester (CFSE; Molecular Probes, Inc., Eugene, OR) cells were washed twice in PBS containing 0.1% BSA and resuspended in this solution at 107 cells/ml. CFSE was t...