expression by these cells, as well as proinflammatory cy-to kines either alone or together with TLR agonists. We first analyzed the impact of agonistic CD40 antibodies on BMEM proliferation and differentiation. The injection of mice with CD40 alone or with TLR4 or TLR9 agonists elicited sub- stantial levels of BMEM and NF B cell proliferation. Despite this proliferation, these BMEM did not differentiate into ASCs. These data are in agreement with our in vitro observations, where CD40 induced the proliferation of, yet failed to di-f ferentiate, poly-BMEM into ASCs. These studies also extend previous in vitro studies where CD40 signaling triggered hu- man BMEM proliferation, but impeded their differentiation into ASCs (Arpin et al., 1995). Using a second approach, we activated T cells in vivo through the injection of agonistic CD3 antibodies, with this treatment given either alone or with TLR agonists. Injection of this antibody is known to elicit rapid and transient activation of T cells, which r-e lease a proinflammatory cytokine storm that includes IL-2, IFN-, and TNF (Ferran et al., 1990; Scott et al., 1990). These stimuli did not induce either BMEM proliferation or differentiation. It is highly likely that the in vivo response of BMEM to overt antibody-mediated CD40 signaling does not represent the hypothetical situation where BMEM and activated T cells transiently engage in a CD40/CD40L synapse independent of an antigen bridge. As we found no BMEM proliferation upon vaccination with an irrelevant thymus-dependent anti- gen or infection with vaccinia virus (a situation generating large numbers of activated CD4 T cells expression CD40L), it is probable that this interaction does not occur and is thus not a mechanism for propagating BMEM renewal. Instead, this data should be interpreted as in vivo proof that CD40 signal- ing drives BMEM proliferation but blocks their differentiation into ASCs, indicating that differentiation occurs upon cessa- tion of CD40 signaling. Immunization with NP-KLH, along with alum and CpG, and infection with vaccinia virus results in the acute induction of both innate and adaptive immunity. BMEM ignored the stimuli associated with these responses and neither proliferated nor differentiated into ASCs during the multi-week period in which these immune responses were occurring. In summary, inflammatory signals associated with TLR signaling, T cell -ac tivation, and adaptive immune responses are incapable of driv- ing BMEM proliferation or differentiation into ASCs. The possibility remains that BMEM and long-lived PCs might survive for the lifetime of the host, with these popula- tions not requiring self-renewal or replenishment and with serological memory not requiring maintenance. However, this hypothesis requires that BMEM and PCs survive for perpe- tuity, and this quality appears unrealistic. From our presented work, we show that BMEM do not clonally expand or diffe-r entiate in response to bystander inflammatory signals. It is possible that there are other signals capable of inducing in vivo BMEM expansion and differentiation that are not included in this analysis. Alternatively, the maintenance of serological memory may occur via a stochastic rather than an induced JEM VOL. 206, August 31, 2009 A RT I C L E process, with BMEM not requiring an extrinsic signal to main- tain serological memory such as those tested herein. MATERIALS AND METHODS Mice and immunizations. These studies were approved by the Institu- tional Animal Care and Use Committee of Dartmouth College (Lebanon, NH). BALB/c mice were purchased from the National Cancer Institute. RAG/ mice were purchased from The Jackson Laboratory. C1-cre mice crossed with Rosa26-EYFP mice have been previously described and were provided by K. Rajewsky (Harvard Medical School, Boston MA) and S. Casola (Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology Foundation, Milan, Italy; Casola et al., 2006). All animals were maintained in a pathogen-free facility at Dartmouth Medical School. For primary immunizations, 10 µg of R-PE (Cyanotech) or 100 µg of NP28- KLH (Biosearch Technologies) adsorbed to prepared alum was injected i.p. in a volume of 200 µl. For secondary challenge, 10 µg of PE (unless indicated otherwise) in PBS in a volume of 200 µl was injected i.p. For VACVWR in- fections, virus was propagated and titered on the 143B cell line as previously described (Fuse et al., 2008), with 2 × 106 PFU injected i.p. For BrdU treat- ment, 0.8 mg/ml was added to the drinking water and changed daily except in Fig. 5 where BrdU was changed every other day. Cell preparation. Single-cell suspensions of lymphocytes were prepared as previously described (Benson et al., 2008). For enrichment of splenocytes for BMEM before phenotyping and BrdU analysis, CD4+, CD8+, IgD+, and IgM+ cells were removed by negative depletion using a mix of biotinylated antibodies. Cells were removed using the EasySep Biotin Selection Kit for Mouse Cells (StemCell Technologies). For enrichment ofMBEM before cell sorting, IgD+ and IgM+ cells were first removed, and then CD43+ cells were removed in a two-step process using the EasySep Mouse B Cell En- richment Kit. Cell culture/reagents. B cells were cultured in RPMI media suppl-e mented with 10% FBS (Atlanta Biologicals), Hepes, 50 µM-ME, and peni- cillin/streptomycin/l-Glutamine. Sorted B cells were cultured in 96-well round-bottomed plates with 10,000–15,000 cells/well and were cultured at 37°C. For in vitro VACVWR CD4 and CD8 recall, BALB/c splenocytes were infected for 2 h with a 10:1 ratio of virus to cells. Infected cells were then plated in a round-bottom 96-well plate with 5 × 160splenocytes har- vested from either D17 VACVWR-infected or noninfected mice added and cultured for 6 h in the presence of 10 µg brefeldin-A and 10 U/ml IL-2. I-n tracellular staining of IFN- was performed as previously described (Fuse et al., 2008). The following reagents and vendors were used in this study: CpG-ODN1826 (5-TCCATGACGTTCCTGACGTT-3) with phos- phorothioate bases (Eurogentec S.A.), LPS 055:B5, BrdU (Sigma-Aldrich), Pam4CSK4, HKLM, poly(I:C), ST-FLA, and FSL1 (Invivogen), S-27609 (3M Pharmaceuticals), IL-4 (Peprotech), CD40 clone FGK45, and CD3 clone 145 2C11 antibodies (Klaus Lube; BioExpress). Flow cytometry. Antibodies against the following antigens were used: B220 (clone 6B2), IgG1 (clone A85-1), IgD clone (11-26c), IgM (clone 11– 41), CD4 (clone GK1.5), CD8 (clone 2.43), IFN- (clone XMG1.2), CD43 (clone 1B11), CD38 (clone 90), CD23 (clone B3B4), CD80 (clone 1610- A1), Ki-67 (clone B56), CD138 (clone 281–2), BrdU (clone PRB-1), CD35 (clone 8C12), GL7, and PNA. NIP-PE and PE-binding cells were detected by staining with NIP20-PE (generated in-house) or with 2 µg/ml PE. For CFSE or DDAO dye dilution, cells were labeled with 5 µM CFSE or 10 µM DDAO in RPMI at 37°C for 10 min (Invitrogen). Intracellular BrdU (BD) and Ki-67 (eBioscience) staining was performed according to the manufac- turers recommendations. Flow cytometry was performed on a refurbished FACSCAN running CellQuest software (BD), with data analysis performed using FlowJo (Tree Star, Inc.). Cell sorts were performed on a FACSAria (BD; Flow Cytometry Facility at Dartmouth Medical School). 2023