Figure 2. BMEM cells undergo rapid self-renewal and differentiation into IgG+ PCs in response to soluble antigen. Either naive or PE-immunized mice were challenged with PBS, 1 µg sPE, or 1 µg sPE + 50 µg CpG and 3 d later, IgM+, and IgG+ PE-PCs from the spleen were quantified by ELISPOT (A and B). To measure the proliferation of BMEM, PE-immunized mice were injected with 5 µg sPE or PBS with BrdU added to the drinking water and con- tinued for five days as shown (C). A group of immunized mice were included that were not given BrdU or sPE (C). At the indicated day after rechallenge, the percentage of PE-BMEM incorporating BrdU was measured by FACS (C). In parallel, IgG+ PE-PCs were quantified in sPE- and PBS-injected mice from both the spleen and BM by ELISpot (D). The levels of IgG+ PE-specific serum antibody titers present on the indicated days after sPE injection were com- pared with titers present in PBS injected mice by ELISA (E). The expression levels of intracellular Ki-67 by PE-BMEM (DumpB220+PE+; pregates not shown; Dump, CD4+CD8+IgD+IgM+) was measured at the indicated time points after sPE injection by FACS, with representative plots depicted (F). Plots were stained and analyzed on the same day. To examine the impact of varying sPE dose during rechallenge, PE-immunized mice were rechallenged with 1, 10, 100 µg sPE or PBS only, with BrdU given daily in the drinking water. After 3 d, the percentage of PE-BMEM incorporating BrdU was measured by FACS (G). The same experimental setup used in G was used in H, except mice were injected with PBS, 1 µg sPE, or 1 µg PE with 50 µg CpG, with BrdU incorporation by PE-BMEM again depicted. Three to five mice per experimental group were used per experiment. Data presented are representative of two to three inde- pendent experiments, with A and B pooled from two independent experiments. Error bars in C–E are expressed as the mean ± SEM. AU, arbitrary units. 2018 IN VIVO ACTIVATION OF MEMORY B CELLS | Benson et al.