A RT I C L E with these BMEM expressing a GL7PNAKi-67hiCD38+ jection with LPS, CpG, or CD3 alone orCD3 and LPS or phenotype. CpG, no proliferation was observed for either NF B cells or To investigate whether antigen-activated BMEM generate PE-BMEM (Fig. 4, A and B, and Fig. S5). These data indicate GCs, GC structures were quantified in spleens where PE- that both NF B cells and PE-BMEM proliferate in response to BMEM were either unchallenged or were responding to sPE or CD40 signals in vivo, whereas TLR agonists or activation sPE + CpG by immunofluorescence (IF) microscopy. As a of T cells withCD3 did not induce BMEM proliferation. positive control for the presence of GCs, naive BALB/c were To determine whether BMEM differentiate into ASCs after immunized with PE/alum and spleens were examined 10 d signaling through CD40, PE-immunized mice were injected later at the height of the GC response. To ensure that PE- with PBS, sPE, CpG, CD40, or both CD40 and CpG. BMEM in the sPE- and sPE + CpG–treated groups and the IgG+ PE-ASCs after agonist injection were quantified, and PE-GC B cells in the D10 PE/alum immunized groups were only in the sPE challenged mice was an increase in IgG+ PE- present and proliferating in these mice, FACS analysis was ASC numbers observed (unpublished data). The numbers of performed on half of the spleens, with BrdU incorporation BM IgG+ PE-ASCs after agonist treatment were also ana- by B220+PE+ cells analyzedF(ig. S4). Upon IF evaluation, in lyzed, with serum harvested on the day of agonist injection the D10 PE/alum immunized spleens, GL7+ clusters were and again when analyzed 14 d later. The only treatment observed and proximal to CD35+ follicular DCs, with these group in which an increase in numbers of BM IgG+ PE- structures detected in 50% of the B cell follicles (Fig. 3, C ASCs and serum PE-specific IgG was observed in the sPE- and D). These GL7+ cell clusters were minimally present in treated cohort (Fig. 4, C and D). the PBS-, sPE-, and sPE + CpG–treated groups (Fig. 3, C These data indicate that TLR4 and TLR9 agonists and and D). Importantly, there was no significant increase in the the proinflammatory cytokine storm elicited by agonistic number of GCs present in spleens where BMEM were acti- CD3 treatment are incapable of activating BMEM and driv- vated by sPE or sPE + CpG in comparison to PBS controls ing their proliferation or differentiation in vivo. Engagement (Fig. 3 D). These data, coupled with the GL7PNAKi- of CD40 (either alone or coupled with TLR4 and TLR9 ag- 67hiCD38+ phenotype displayed by BMEM after in vivo anti- onists) drives both NF B cell and BMEM proliferation, but is gen-dependent activation, demonstrate that GCs are not incapable of mediating their differentiation into ASCs. created by BMEM after antigen rechallenge. Analysis of the in vivo BMEM cell response to TLR agonists, CD40 agonists, and bystander T cell help The in vivo BMEM response to TLR agonists, CD40 agonists, and bystander T cell help was determined. PE-immunized mice were injected with PBS, sPE as a positive control, com- binations of TLR agonists, agonistic CD40, and/or CD3. We treated mice with agonist doses at or greater than doses known to have an optimal biological effect in vivo (Ferran et al., 1990; Ahonen et al., 2004). In data where 50 µg of CpG was used, similar data were obtained using 100 µg (unpub- lished data). Furthermore, the efficacy of the TLR agonists was confirmed by observing an induction in the expression of CD86 by both NF B cells and CD11c+ DCs 24 h after TLR agonist injection in vivo (unpublished data). PE-BMEM prolif- eration was tracked by BrdU incorporation over a period of 3 d (Fig. S5 A). NF B cells were included in this analysis and were identified as B220+IgD+ cells (Fig. S5 B). Injection with PBS alone elicited very low amounts of BrdU incorporation by both NF B cells and PE-BMEM, whereas injection with sPE elicited only BMEM proliferation (Figs. 4, A and B, and Fig. S5). It was observed that both BMEM and NF B cells pro- liferate in response to injection with agonistic CD40 anti- body alone or coupled with LPS or CpG, with the mean proliferation of both subsets as measured by BrdU incorpora- tion between 25 and 50% (Fig. 4, A and B, Fig. S5). After in- BMEM cells do not proliferate or differentiate into ASCs in vivo after immunization with an irrelevant protein antigen or vaccinia virus infection The capacity of PE-BMEM to proliferate and differentiate in response to either immunization with NP-KLH/alum ad- ministered with CpG or infection with vaccinia virus West- ern Reserve strain (VACVWR) was analyzed. The levels of BMEM proliferation and their differentiation into ASCs were measured 17 d later with this time chosen to allow for the full development of an adaptive immune response against either NP-KLH or VACVWR. After immunization with VACVWR or NP-KLH, PE-BMEM did not incorporate any more BrdU (15.9% ± SEM 1.8 and 16.6% ± SEM 2.3, respectively) and then after injection with PBS alone (19.22% ± SEM 1.5;Fig. S6 C and Fig. 5 A). As anticipated, sPE induced robust PE- BMEM cell proliferation, with the majority (73% ± SEM 2.8) of cells BrdU+ (Fig. S6 C and Fig. 5 A). The generation of IgG+ PE-ASCs was also measured by comparing the levels of PE+ IgG+ serum antibody titers present before and after im- munization and by quantifying the number of IgG+ PE-ASCs present in the spleen and BM by ELISPOT. VACVWR and NP-KLH immunizations did not impact the number of IgG+ PE-ASCs present in either the spleen or the BM 17 d after immunization (Fig. 5, B and C). There was also no difference in the levels of IgG+ PE-specific serum antibody titers after VACVWR or NP-KLH immunization (Fig. 5 D). Consistent 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 com- parisons between the indicated experimental groups: ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. JEM VOL. 206, August 31, 2009 2019