A RT I C L E rechallenge, BMEM began to proliferate, with this response The observation that 50% of BMEM respond to sPE as peaking by day 4 (Fig. 2 C and Fig. S3 A). In mice analyzed indicated by BrdU incorporation (Fig. 2 C and Fig. S3 A) led during the BrdU chase period (days 11 and 24 after sPE rechal- us to question whether either the antigen dose or the absence lenge), a proportion of BMEM remained BrdU+ at a percentage of adjuvant during antigen administration was limiting the similar to that observed at the peak of the response (35.5% ± frequency of responding BMEM. To test this, we varied the SEM 4.6 and 38.4% ± SEM 5.1 versus 48.9% ± SEM 5.8, re- amount of sPE injected or added CpG during antigen rechal- spectively), indicating that 71–77% of BMEM proliferation oc- lenge, with BrdU administration concurrently initiated. curs within the first 5 d after antigen rechallenge (Fig. 2 C and When analyzed 3 d later, it was observed that the dose of an- Fig. S3 A). A second PE-binding population was also observed tigen positively correlated with the percentage of prolifera-t in these experiments, with this population displaying a ing BMEM (Fig. 2 G and Fig. S3 B). The addition of CpG had B220PE+ phenotype (Fig. S3 A). When the BrdU incorpora- no further impact on the frequency of responding PE-BMEM tion frequencies by the B220PE+ population in PBS versus (Fig. 2 G and Fig. S3 C). However, the addition of CpG to sPE-immunized mice were compared, no differences were sPE modestly increased the number of splenic IgG+ PE-ASCs observed between the two groups over the 24-d kinetic elicited after 3 d when compared with sPE alone (Fig. 2 B). analysis (unpublished data). This indicates that the B220PE+ In sum, these data indicate that antigen can be a limiting fac- population does not respond to sPE, and as antigen-respon- tor in dictating the frequency of responding BMEM. Notably, siveness is a crucial element of adaptive memory, we excluded TLR9 agonists failed to enhance the frequency of activated these cells from further analysis. As BrdU labeling measures BMEM. This point indicates that CpGs alone may not play a the accumulation of proliferation over the time of the BrdU role in driving in vivo BMEM activation. “pulse” period, we also analyzed BMEM expression of Ki-67 at various times after antigen challenge (Fig. 2 F). Ki-67 expre-s BMEM cells proliferate upon in vivo antigen encounter sion is an indicator of all stages of the cell cycle and provides a and do not express GC markers or generate GC structures snapshot of the proliferative state of the BMEM at a given point The rapid kinetics of the BMEM response led us to question in time (Gerdes et al., 1984). PE-BMEM are almost uniformly whether BMEM form GCs after antigen encounter. Clonally Ki-67, which is indicative of their quiescent state (Fig. 2 F). expanding BMEM were tracked to define their transitional By days 3 and 4 after sPE rechallenge, 76–77% of BMEM cells stages en route to differentiating into ASCs. Under steady- were Ki-67+. At day 5, Ki-67 expression began to dissipate, state conditions, PE-BMEM displayed a GL7PNAKi- and most PE-BMEM exhibited decreased Ki-67 expression on 67CD38+ phenotype as anticipated (Fig. 3 A; Lalor et al., day 7, with this decrease more pronounced by day 11. By day 1992). As a positive control for GC markers, Peyer’s patches 24, PE-BMEM were once again uniform in their lack of Ki-67 were harvested and gated on B220+IgD cells, a population expression (Fig. 2 F). In correlating the expression pattern of known to be primarily GC B cells, with these cells exhibiting Ki-67 by BMEM (Fig. 2 F) with the 5-d BrdU pulse followed by a GL7+PNA+Ki-67hiCD38 phenotype (Butcher et al., a chase period (Fig. 2 C), the results indicate that the majority 1982). When PE-BMEM were harvested and analyzed 3 d after of BMEM proliferation occurs between days 2 and 5 after sPE sPE challenge, they were GL7PNAKi-67hiCD38+, indi- rechallenge. In addition, the presence of BrdU+ BMEM 24 d af- cating these cells had responded to antigen because of Ki-67 ter sPE injection provides direct evidence that BMEM generate expression, yet failed to express the GC markers GL7 and daughter BMEM after secondary antigen encounter. PNA (Fig. 3 A). It was plausible that the failure of antigen- A rapid increase in the numbers of IgG+ PE-ASCs in responding BMEM to gain a GC phenotype was caused by the both the spleen and BM was observed 3 d after sPE rechal- solitary use of sPE, and that the induction of a GC phenotype lenge (Fig. 2, B and D). This burst in ASC numbers peaked requires the presence of an adjuvant. Therefore, CpG was 4 d after challenge in the spleen and 5 d in the BM, after added to sPE, and 3 d later, the phenotype of the responding which ASC numbers gradually subsided over the next 20 d BMEM was analyzed. The addition of CpG to sPE again elic- (Fig. 2 D). Corresponding to the increase of ASCs after re- ited GL7PNAKi-67hiCD38+ BMEM (Fig. 3 A). It was con- challenge was an increase in anti-PE IgG serum titers increa-s sidered possible that BMEM gain a GC phenotype later than 3 d ing 10-fold over the next 20 d (Fig. 2 E). These data show after antigen encounter, so the phenotype of BMEM was ana- that ASCs are rapidly generated within 3 d after antigen re- lyzed at later points in time. The results indicate that PE-BMEM challenge and are present to substantial degrees in both the maintain a GL7PNACD38+ phenotype throughout the spleen and the BM. Soon thereafter, ASC numbers declined, cell cycle (Fig. 3 B). These data define a proliferating state indicating that short-lived ASCs are present in both organs. of BMEM occurring shortly after in vivo antigen encounter, (CD40), CD40, 10 µg/ml; and IL-4, 10 ng/ml. The percentage of B220intCD138+ cells derived from NF B cells (open bars) or BMEM (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 BMEM are pooled from six to eight mice per experiment, with NF B cells isolated from one mouse per ex- periment. Results in G–I are expressed as the mean ± SEM. JEM VOL. 206, August 31, 2009 2017