Figure 3. BMEM cells proliferate upon in vivo antigen encounter and do not express GC markers or generate GC structures. PE-immunized mice were injected with PBS, 10 µg sPE, or 10 µg sPE + 50 µg CpG. 3 d later, PE-BMEM (Dump-B220+PE+; pregates not shown and Dump = CD4+CD8+IgD+IgM+) in each treatment group were analyzed for their expression of GL7, PNA, Ki-67, and CD38 by FACS. Included in this analysis were lymphocytes harvested from the Peyer’s patches of the same mice and gated on B220+IgD (PP-GC; pregates not depicted). Representative data depicted are histograms of indicated phenotypic marker (A). A similar experiment was performed with responding PE-BMEM examined at various time points after 10 µg sPE rechallenge (B). To detect the presence of GCs in mice harboring antigen-responding BMEM, PE-immunized mice were rechallenged with PBS, sPE, or sPE + CpG. Included in the analysis were BALB/c mice that had received a primary PE/alum immunization 10 d prior. GCs in each group were quan- tified by IF microscopy (C) and are represented as the percentage of splenic IgD+ B cell follicles harboring GL7+ foci, with 50–75 follicles counted/spleen (D). A and B are representative of three experiments, with three to four mice analyzed on each day. The IF experiment contained three mice analyzed from two independent experiments (C and D). For IF images, green represents GL7, blue represents IgD, and purple represents CD35. Bar, 100 µm. In D, each point represents an independent mouse, with line representing the mean. Results of an unpaired Student’s t test in D are shown as a comparison between indicated groups. ***, P < 0.001. 2020 IN VIVO ACTIVATION OF MEMORY B CELLS | Benson et al.