1997; McHeyzer-Williams et al., 2006). Shortly after r-e agonists, polyclonal T cell activation, protein vaccination, call, BMEM cells are once again present in their initial an-a and even acute vaccinia virus infection, BMEM neither clon- tomical positions (Liu et al., 1991). Despite numerous ally expand nor differentiate into ASCs, and thus appear ig- studies carefully mapping out the events of the primary norant to overt bystander inflammatory signals. humoral immune response toward protein antigens (Jacob et al., 1991; MacLennan, 1994; Allen et al., 2007), re-la RESULTS tively little is known about the events occurring during a Antigen-specific and polyclonal BMEM cells are responsive secondary humoral immune response, including whether to TLR agonists in vitro and display an enhanced capacity antigen reencounter results in the expression of GC mark- to differentiate into ASCs when compared with NF B cells ers by antigen-responding BMEM and/or if BMEM form GC A comprehensive analysis of BMEM responsiveness to antigen structures en route to differentiating into PCs (Liu et al., and bystander inflammatory stimuli was undertaken both in 1991; MacLennan, 1994; McHeyzer-Williams et al., 2006). vitro and in vivo. The term “bystander” is defined herein as As the fate and behavior of antigen-activated BMEM is criti- stimuli acting on BMEM independent of BCR engagement. cal to our understanding of BMEM self-renewal, included in In these studies, we tracked BMEM specific for the thymus- this study is a detailed analysis of the in vivo response of dependent protein antigen R-phycoerythrin (PE), as immu- BMEM toward cognate antigen. nization of BALB/c mice with this fluorophore elicits a The mechanisms mediating the survival of BMEM are un- detectable population of PE-binding BMEM at a high resolu- known. Although BMEM require phospholipase C  signaling tion (referred to as PE-BMEM; Hayakawa et al., 1987). After through the B cell receptor (BCR) for survival, it is clear that immunization with PE adsorbed to alum i.p., PE-BMEM are the BMEM BCR need not be permanently engaged with anti- readily detectable in the spleen at 0.01–0.05% of total splen-o gen, as BMEM do not require the presence of persisting anti- cytes beginning 21 d after immunization, with this popula- gen or antigen complexes to survive (Maruyama et al., 2000; tion still present 60 d after priming F(ig. S1; Hayakawa et al., Anderson et al., 2006; Hikida et al., 2009). It is apparent that 1987; Schittek and Rajewsky, 1990; Benson et al., 2008). In BMEM occupy a survival niche independent from other B-2 nonimmunized mice, a minute naive PE-binding population lineage cell subsets, as, unlike all other mature cells of the B-2 is observed at a 10-fold lower frequency (Fig. S1). The PE- lineage, they do not require BAFF and APRIL survival sig- BMEM in immunized mice are predominantly B220+PE+CD3 nals (Benson et al., 2008; Scholz et al., 2008). At the level of 8+IgDIgG1+CD80hi and express a phenotype consistent the organism, it is thought that humoral memory as a whole with long-lived affinity-matured BMEM (Fig. S1; Anderson is sustained by the stem cell–like qualities of MBEM, with this et al., 2007). In all experiments (unless indicated otherwise), cell type undergoing low levels of self-renewal and replen-ish PE-BMEM from PE/alum-immunized mice were analyzed ment of the long-lived PC pool over the lifetime of the o-r between days 60 and 90 after primary immunization. ganism (Traggiai et al., 2003). To sustain serological memory The response of PE-BMEM to inflammatory stimuli was in the absence of cognate antigen, it has been hypothesized first analyzed by examining the response profile of BMEM to a that quiescent BMEM are periodically activated by Toll-like panel of TLR agonists in vitro. We also included two other receptor (TLR) agonists or bystander T cell help to undergo sources of BMEM in these in vitro analyses to first ensure that self-renewal events and differentiate into antibody-secreting the results were reproducible in multiple systems and to test cells (ASCs) such as PBs and PCs in an antigen-independent whether BMEM activation occurs in the absence of BCR manner (Bernasconi et al., 2002). A crucial element of this cross-linking. The second system used BMEM raised against theory was the prediction that inflammatory stimuli assoc-i and specific for the hapten 4-hydroxy-3-nitrophenylacetyl ated with adaptive immune responses drive the activation of (NP), with this system having been previously characterized BMEM in an antigen-independent (i.e., nonspecific) manner (McHeyzer-Williams et al., 1991; Lalor et al., 1992). The in vivo (Bernasconi et al., 2002, 2003; Traggiai et al., 2003). third system used BMEM from a transgenic mouse in which Whether BMEM are indeed reactive to bystander inflamma- the polyclonal BMEM population is readily identified. This la-t tory stimuli in vivo has not yet been tested from the vantage ter source allowed for the isolation of BMEM without the need point of BMEM. for BCR engagement by antigen, as when PE-BMEM and In this work, we detail the ability of BMEM to undergo NP-BMEM are purified using antigen binding to denote speci- self-renewal and to differentiate into ASCs in response to ficity, cross-linking the BCR is inevitable. both antigen-dependent and bystander inflammatory stimuli. In studying these events, we also demonstrate that during PE-BMEM cells. From PE-immunized mice, PE-BMEM clonal expansion in response to cognate antigen, BMEM do were first enriched by negative selection, and then elec- not express a GC B cell phenotype, and that GC structures tronically sorted based on a B220+PE+dumpCD38+ phe- are not generated en route to differentiating into ASCs. We notype, with post-sort analysis indicating high levels of find that BMEM are responsive to inflammatory stimuli in v-i purity (Fig. S2 A). tro, with activated BMEM displaying a markedly enhanced ca- pacity to differentiate into ASCs when compared with naive NP-specific BMEM cells. NP-BMEM were generated in a follicular (NF) B cells. However, in vivo, in response to TLR transgenic mouse strain where Cre recombinase was inserted 2014 IN VIVO ACTIVATION OF MEMORY B CELLS | Benson et al.