with our earlier observations (Fig. 2, D and E), sPE induced a substantial and significant increase in the number of IgG+ PE-ASCs in both the spleen and BM (Fig. 5, B and C) and led to an 30-fold increase in the levels of IgG+ PE-specific serum antibody titers (Fig. 5 D). Both cellular and humoral anti-VACVWR immune responses were verified to have oc- curred by testing for the presence of IFN-+ VACVWR- specific CD4 and CD8 T cells in spleens harvested from VACVWR-infected mice, but not naive mice, and by observ- ing for the presence of anti-VACVWR IgG serum antibodies only in infected mice (Fig. S6, A and B). A RT I C L E These data collectively demonstrate that immunization with NP-KLH and VACVWR infection does not induce PE- BMEM proliferation or differentiation into ASCs. Thus, BMEM are unresponsive to stimuli generated during a bystander im- mune response. DISCUSSION A detailed analysis of the in vivo secondary immune response of BMEM in response to cognate antigen versus noncognate inflammatory bystander signals is presented. These data show that BMEM respond robustly upon rechallenge with cognate antigen, but do not acquire the expression of GC markers or form GCs in response to cognate antigen. In striking contrast, the response of BMEM to a battery of proinflammatory signals is remarkably silent. It has been proposed that serological Figure 4. Analysis of the in vivo BMEM cell response to TLR ago- nists, 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 CD40, or 5 µg CD3 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-BMEM incorporating BrdU was analyzed 3 d later by FACS (A and B, as indicated). To analyze whether CpG and CD40 either alone or in combination enhanced the number of PE-PCs in the BM, mice re- ceived the indicated agonist and were sacrificed 14 d later, with the num- ber 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 anti- body 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 indi- vidual mouse, with mean indicated by horizontal line. In D, each group contains seven mice, with data expressed as the mean ± SEM. AU, arbi- trary 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. JEM VOL. 206, August 31, 2009 Figure 5. BMEM cells do not proliferate or differentiate into PCs in vivo after immunization with an irrelevant protein antigen or vac- cinia virus infection. PE-immunized mice were injected i.p. with PBS, 10 µg sPE, 2 × 106 PFU VACVWR, 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- BMEM–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 experi- ments. Results of an unpaired Student’s t test are shown as comparisons between the indicated experimental groups in A–C. ***, P < 0.001. 2021