memory is sustained by BMEM responding to bystander inflam- mental system excluded testing whether IgM+ BMEM are re- matory signals by undergoing proliferation and differentia- sponsive to bystander inflammatory signals. There is a growing tion. However, the findings of this study indicate that BMEM body of evidence both in favor of and in conflict with the ignore these signals (TLR agonists, polyclonal T cell acti-va hypothesis that serological memory is sustained by the activa- tion, aggressive vaccination, and acute viral infection), as they tion of BMEM by bystander inflammation. Evidence in favor fail to drive BMEM proliferation or differentiation in vivo. of this theory includes in vitro observations in which human Despite the preponderance of studies detailing the B cell BMEM were shown to have increased sensitivity and to selec- dynamics of the primary humoral immune response, surpris- tively proliferate and differentiate into ASCs in response to ingly few studies have detailed the events of the secondary bystander stimuli as compared with NF B cells (Bernasconi humoral immune response. We have tracked BMEM and dem- et al., 2002, 2003). In vivo studies in humans found that for onstrate that cognate antigen drives the rapid clonal expan- single-pathogen specificities, serum antibody levels linearly sion of BMEM and their terminal differentiation into ASCs, correlated with the frequency of BMEM present in a host, in- with these events occurring in the absence of GC formation. dicating that the ASC compartment was genealogically linked As has been widely reported, BMEM rapidly differentiate to to and undergoing replenishment by the BMEM compartment ASCs upon secondary antigen encounter (Höfer et al., 2006; (Bernasconi et al., 2002). Mouse studies also supported this Minges Wols et al., 2007), with this ASC response first de- link between the BMEM and ASC compartments as elimina- tectable 3 d after antigen challenge in both the spleen and the tion of virus-specific BMEM led to a slow decay of virus- BM and with the splenic ASC response an order of magni- specific ASC numbers over time in comparison to mice still tude greater than that observed in the BM. This wave of containing BMEM (Slifka et al., 1998). Lastly, it was observed ASCs dissipates after 11 d, and it is probable that the remain- that during an immune response against a single pathogen, an ing ASCs in the BM 24 d after antigen challenge have been increase in both blood ASC numbers and serum antibody culled into the long-lived PC pool (Manz et al., 1997; Höfer levels occurred for specificities to other pathogens (Bernasconi et al., 2006; Minges Wols et al., 2007). These data detail the et al., 2002). This result was interpreted as evidence that host activation of BMEM and define their proliferation as occurring BMEM were recipients of antigen-independent stimuli and re- between days 2 and 5 after antigen encounter. Proliferating sponded by differentiating into ASCs. BMEM occupy a transitional state defined as antigen-binding Despite these data suggesting that BMEM respond to by- B220+CD38+Ki-67+GL7PNA before generating quies- stander inflammation, more recent studies have put this hy- cent daughter BMEM and/or differentiating into ASCs. This pothesis under scrutiny. In particular, further attempts to blasting state is unique because of the presence of high levels correlate antigen-specific BMEM numbers with circulating ASCs of CD38 expression by proliferating cells; CD38 is a phen-o in human subjects have been unsuccessful (Amanna et al., typic marker previously used to denote quiescent NF B cells 2007). Mouse studies where long-lived ASC numbers were and BMEM and is lost upon B cell activation and entry into the quantified after BMEM depletion observed no decay in ASC GC (Oliver et al., 1997). Our finding that BMEM cells do not numbers over time, contrary to initial reports (Ahuja et al., form or enter a GC reaction during their differentiation and 2008; DiLillo et al., 2008). Lastly, a series of studies reported expansion is consistent with classical studies indicating that that in mice that were administered a TLR agonist or in hu- BMEM might not undergo further SHM and isotype switching mans that were given a vaccine, ASC numbers and serum anti- upon rechallenge with antigen (Askonas et al., 1970; Okumura body levels against specificities other than the immunizing et al., 1976; Siekevitz et al., 1987). In these studies, limiting antigen did not increase (Di Genova et al., 2006; Amanna numbers of hapten-specific “parental” BMEM were adoptively et al., 2007; Xiang et al., 2007; Richard et al., 2008). transferred together with carrier-primed T cells into lightly Our studies show that the in vitro sensitivities of BMEM to irradiated hosts, followed by immunization of the hosts activation are not representative of their sensitivities in vivo with soluble hapten-carrier antigen. Upon analyzing the and cast doubt on the hypothesis that BMEM can be activated isotype distribution and SHM exhibited by the parental by bystander inflammation. Our in vitro studies examining BMEM and their progeny generated in response to cognate the response profiles of murine BMEM to both antigen-depen- antigen, no change in diversity in either measure was ob- dent and -independent stimuli extended prior studies using served. Our data showing that BMEM neither gain GC mark- human (Arpin et al., 1997; Bernasconi et al., 2002) and mu- ers nor seed GCs are in concordance with these studies. It rine (Richard et al., 2008) BMEM, as we observe that murine is apparent that BMEM, at least in these studies and in our BMEM are sensitive to TLR agonists and have an enhanced presented data, are not instructed to enhance the affinity or intrinsic ability to differentiate into ASCs upon activation isotype distribution of their antigen receptors during sec- with TLR agonists, regardless of whether the BCR is en- ondary antigen encounter. gaged by antigen. Given these data, and our knowledge of Our data indicate that quiescent IgG+ BMEM are not acti- the robust response of BMEM to cognate antigen in vivo, an vated by TLR agonists or bystander T cell help, and thus do exploration of the response of BMEM to several nonspecific not undergo self-renewal events or differentiate into ASCs in inflammatory signals in vivo demonstrated that BMEM are in an antigen-independent manner. We should note that our fact unresponsive to these stimuli. The first panel of signals analysis was limited to IgG+ PE-BMEM and that our experi- tested was derived from activated T cells and included CD154 2022 IN VIVO ACTIVATION OF MEMORY B CELLS | Benson et al.