B_MEM cells do not proliferate or differentiate into PCs in vivo after immunization with an irrelevant protein antigen or vaccinia virus infection. PE-immunized mice were injected i.p. with PBS, 10 µg sPE, 2 × 10⁶ PFU VACV_WR, or 100 µg NP-KLH/alum + 50 µg CpG, as indicated. BrdU was added to the drinking water at the time of injection and for the duration of the experiment. After 17 d, the percentage of PE-B_MEM–incorporating BrdU was quantified by FACS analysis (A). On the same day, the numbers of IgG⁺ PE-PCs were quantified in both the spleen and BM for each treatment group by ELISpot (B and C, respectively). Serum was harvested 3 d before immunization and again on day 17, with IgG⁺ PE-specific serum antibody titers from both time points shown (D). Horizontal lines represent the mean (A–C). In A–C, three to four mice per experimental group were used per experiment. Data shown in 5A is pooled from two independent experiments, and data shown in B and C is representative of one of these experiments. Results in D are expressed as the mean ± SEM. AU, arbitrary units. Seven to eight mice were used in each treatment group. D is the pooled data of two independent experiments. Results of an unpaired Student's t test are shown as comparisons between the indicated experimental groups in A–C. ***, P < 0.001.

B_MEM 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-B_MEM (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-B_MEM examined at various time points after 10 µg sPE rechallenge (B). To detect the presence of GCs in mice harboring antigen-responding B_MEM, 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 quantified 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.

Analysis of the in vivo B_MEM cell response to TLR agonists, CD40 agonists, and bystander T cell help. PE-immunized mice were injected i.p. with PBS, 10 µg sPE, 50 µg CpG, 50 µg LPS, 100 µg αCD40, or 5 µg αCD3 either alone or in combination as indicated. For A and B, BrdU was added to the drinking water at the time of injection and for the duration of the experiment. The percentage of splenic NF B cells and PE-B_MEM incorporating BrdU was analyzed 3 d later by FACS (A and B, as indicated). To analyze whether CpG and αCD40 either alone or in combination enhanced the number of PE-PCs in the BM, mice received the indicated agonist and were sacrificed 14 d later, with the number of IgG⁺ PE-PCs in the BM quantified by ELISpot (C). Furthermore, serum was harvested on the same day as agonist injection (Day 0), as well as on the day of sacrifice (Day 14), with the titers of IgG⁺ PE-specific antibody titers analyzed on both days by ELISA (D). In A–C, three to four mice per experimental group were used per experiment. Data shown is pooled from three independent experiments. In A–C, each dot depicts an individual mouse, with mean indicated by horizontal line. In D, each group contains seven mice, with data expressed as the mean ± SEM. AU, arbitrary units. D is the pooled data of two independent experiments. Results of an unpaired Student's t test are shown as comparisons between the indicated experimental group and the PBS control group: no denotation, no significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Antigen-specific and polyclonal B_MEM cells are responsive to TLR agonists in vitro and display an enhanced capacity to differentiate into ASCs compared with NF B cells. NP-B_MEM and PE-B_MEM were sorted by FACS, along with NF B cells. NP-B_MEM and NF B cells were stained with DDAO and PE-B_MEM, and NF B cells were stained with CFSE. Cells were cultured for 3 d with 10 µg/ml LPS, whereupon the cell surface phenotype was analyzed for the expression of B220 and CD138 (A) or DDAO or CFSE (B), with the percentage of B220⁺CD138⁺ cells depicted (A). (B) Filled histograms, B cells + IL-4; gray lines, B_MEM; black lines, NF B cells. To analyze the reactivity of B_MEM to TLR agonists in the absence of BCR cross-linking, poly-B_MEM were used. Spleens from NP-KLH–immunized Cγ1-cre/ROSA-YFP mice were enriched for B_MEM, with poly-B_MEM present in the remaining population sorted by the phenotype B220⁺IgG1/YFP⁺CD38⁺Dump⁻CD80⁺. Dump gates, CD43⁺IgD⁺IgM⁺ cells (C). Both poly-B_MEM and NF B cells were labeled with DDAO and cultured for 3 d in the presence of either 10 µg/ml LPS or 5 µM CpG, whereupon the cell surface phenotype was analyzed for the expression of B220 and CD138 (D) and DDAO dye dilution (E). (E) Gray lines, B cells + IL-4; black lines, NF or poly-B_MEM as indicated. Proliferation as a function of CD138 expression is shown, with the percentage of cells that have diluted DDAO and are expressing CD138 shown (F). The ability of cells derived from either NF B cells (open bars) or B_MEM (filled bars) after 3 d of LPS culture to secrete antibody of the IgM, IgG1, IgG2a, IgG2b, IgG3, IgE, and IgA isotypes was analyzed by ELISpot analysis (F). The response profiles of poly-B_MEM and NF B cells to a panel of TLR agonists was examined after 3 d of culture with the indicated agonist. The agonists used, their abbreviations, the receptors for which they are specific for, and the concentrations used are as follows: Pam3CSK4 (P3C), TLR1/2, 1 µg/ml; LPS (LPS), TLR4, 10 µg/ml; FSL-1 (FSL1), TLR6/2, 1 µg/ml; S-27609 (609), TLR7, 1 µg/ml; CpG ODN 1826 (CpG), TLR9, 5 µM, αCD40 (αCD40), CD40, 10 µg/ml; and IL-4, 10 ng/ml. The percentage of B220^intCD138⁺ cells derived from NF B cells (open bars) or B_MEM (filled bars; H) and the percentage of cells proliferating as measured by dye dilution of DDAO (I) are depicted. Data presented is representative (A–G) or pooled (H and I) from three to five independent experiments. Purified B_MEM are pooled from six to eight mice per experiment, with NF B cells isolated from one mouse per experiment. Results in G–I are expressed as the mean ± SEM.

B_MEM 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 B_MEM, PE-immunized mice were injected with 5 µg sPE or PBS with BrdU added to the drinking water and continued 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-B_MEM 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 compared with titers present in PBS injected mice by ELISA (E). The expression levels of intracellular Ki-67 by PE-B_MEM (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-B_MEM 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-B_MEM again depicted. Three to five mice per experimental group were used per experiment. Data presented are representative of two to three independent experiments, with A and B pooled from two independent experiments. Error bars in C–E are expressed as the mean ± SEM. AU, arbitrary units. Each point in A, B, G, and H represents an individual mouse, with horizontal line representing the mean. Results of a Student's t test are shown as comparisons between the indicated experimental groups: ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001.