The hypothesis that bystander inflammatory signals promote memory B cell (BMEM) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220+IgG+ BMEM toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent BMEM clonally expand. Surprisingly, proliferating BMEM do not acquire germinal center (GC) B cell markers before generating daughter BMEM and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of BMEM proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of BMEM occurred. The absence of a BMEM response to nonspecific inflammatory signals clearly shows that BMEM proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.

Life-long immunity is central to the survival of the host. This immunity is engendered by the persistence of memory T cells, memory B cells (BMEM), and long-lived BM plasma cells (PCs; Amanna et al., 2007; Dörner and Radbruch, 2007). Long-lasting B cell–mediated immunity has been referred to as serological memory (Traggiai et al., 2003) and can be sustained by recurrent antigen exposure. In the absence of periodic exposure to antigen, it is thought that the production of inflammatory signals to unrelated antigens serve as mediators sustaining serologic memory through activation of BMEM in a noncognate manner. The in vitro and in vivo response profiles of antigen-specific BMEM to cognate antigen and “nonspecific” or bystander inflammatory mediators are analyzed herein to critically evaluate the mechanisms underlying the maintenance of serological memory.

BMEM and long-lived PCs are the products of the germinal center (GC) reaction and express somatically hypermutated (SHM) immunoglobulin receptors of the switched isotypes (MacLennan, 1994; McHeyzer-Williams and Ahmed, 1999). After BMEM exit from the GC reaction, they colonize the splenic marginal zones (MZs) and the B cell follicles, where they reside in a quiescent state for months or, likely, years (Liu et al., 1988, 1991 ,Schittek and Rajewsky, 1990; Anderson et al., 2007). Upon rechallenge with cognate antigen, histological studies in rats observed that BMEM egress toward the T cell–rich periarteriolar lymphocytic sheath and, upon receiving T cell help, proliferate and differentiate into plasmablasts (PBs) situated in both the periarteriolar lymphocytic sheath and in the splenic red pulp (Liu et al., 1991). Kinetic studies indicate that the generation of this response is rapid and peaks ∼4-5 d after secondary antigen encounter (Liu et al., 1991; McHeyzer-Williams et al., 2000; Minges Wols et al., 2007). The PB response dissipates shortly after its initiation, with a subset of PBs further differentiating into long-lived PCs resident in the BM (Liu et al., 1991; Manz et al., 1997; McHeyzer-Williams et al., 2006). Shortly after recall, BMEM cells are once again present in their initial anatomical positions (Liu et al., 1991). Despite numerous studies carefully mapping out the events of the primary humoral immune response toward protein antigens (Jacob et al., 1991; MacLennan, 1994; Allen et al., 2007), relatively little is known about the events occurring during a secondary humoral immune response, including whether antigen reencounter results in the expression of GC markers by antigen-responding BMEM and/or if BMEM form GC structures en route to differentiating into PCs (Liu et al., 1991; MacLennan, 1994; McHeyzer-Williams et al., 2006). As the fate and behavior of antigen-activated BMEM is critical to our understanding of BMEM self-renewal, included in this study is a detailed analysis of the in vivo response of BMEM toward cognate antigen.

The mechanisms mediating the survival of BMEM are unknown. Although BMEM require phospholipase C γ signaling through the B cell receptor (BCR) for survival, it is clear that the BMEM BCR need not be permanently engaged with antigen, as BMEM do not require the presence of persisting antigen or antigen complexes to survive (Maruyama et al., 2000; Anderson et al., 2006; Hikida et al., 2009). It is apparent that BMEM occupy a survival niche independent from other B-2 lineage cell subsets, as, unlike all other mature cells of the B-2 lineage, they do not require BAFF and APRIL survival signals (Benson et al., 2008; Scholz et al., 2008). At the level of the organism, it is thought that humoral memory as a whole is sustained by the stem cell–like qualities of BMEM, with this cell type undergoing low levels of self-renewal and replenishment of the long-lived PC pool over the lifetime of the organism (Traggiai et al., 2003). To sustain serological memory in the absence of cognate antigen, it has been hypothesized that quiescent BMEM are periodically activated by Toll-like receptor (TLR) agonists or bystander T cell help to undergo self-renewal events and differentiate into antibody-secreting cells (ASCs) such as PBs and PCs in an antigen-independent manner (Bernasconi et al., 2002). A crucial element of this theory was the prediction that inflammatory stimuli associated with adaptive immune responses drive the activation of BMEM in an antigen-independent (i.e., nonspecific) manner in vivo (Bernasconi et al., 2002, 2003; Traggiai et al., 2003). Whether BMEM are indeed reactive to bystander inflammatory stimuli in vivo has not yet been tested from the vantage point of BMEM.

In this work, we detail the ability of BMEM to undergo self-renewal and to differentiate into ASCs in response to both antigen-dependent and bystander inflammatory stimuli. In studying these events, we also demonstrate that during clonal expansion in response to cognate antigen, BMEM do not express a GC B cell phenotype, and that GC structures are not generated en route to differentiating into ASCs. We find that BMEM are responsive to inflammatory stimuli in vitro, with activated BMEM displaying a markedly enhanced capacity to differentiate into ASCs when compared with naive follicular (NF) B cells. However, in vivo, in response to TLR agonists, polyclonal T cell activation, protein vaccination, and even acute vaccinia virus infection, BMEM neither clonally expand nor differentiate into ASCs, and thus appear ignorant to overt bystander inflammatory signals.

RESULTS

Antigen-specific and polyclonal BMEM cells are responsive to TLR agonists in vitro and display an enhanced capacity to differentiate into ASCs when compared with NF B cells

A comprehensive analysis of BMEM responsiveness to antigen and bystander inflammatory stimuli was undertaken both in vitro and in vivo. The term “bystander” is defined herein as stimuli acting on BMEM independent of BCR engagement. In these studies, we tracked BMEM specific for the thymus-dependent protein antigen R-phycoerythrin (PE), as immunization of BALB/c mice with this fluorophore elicits a detectable population of PE-binding BMEM at a high resolution (referred to as PE-BMEM; Hayakawa et al., 1987). After immunization with PE adsorbed to alum i.p., PE-BMEM are readily detectable in the spleen at 0.01–0.05% of total splenocytes beginning 21 d after immunization, with this population still present 60 d after priming (Fig. S1; Hayakawa et al., 1987; Schittek and Rajewsky, 1990; Benson et al., 2008). In nonimmunized mice, a minute naive PE-binding population is observed at a 10-fold lower frequency (Fig. S1). The PE-BMEM in immunized mice are predominantly B220+PE+CD38+IgDIgG1+CD80hi and express a phenotype consistent with long-lived affinity-matured BMEM (Fig. S1; Anderson et al., 2007). In all experiments (unless indicated otherwise), PE-BMEM from PE/alum-immunized mice were analyzed between days 60 and 90 after primary immunization.

The response of PE-BMEM to inflammatory stimuli was first analyzed by examining the response profile of BMEM to a panel of TLR agonists in vitro. We also included two other sources of BMEM in these in vitro analyses to first ensure that the results were reproducible in multiple systems and to test whether BMEM activation occurs in the absence of BCR cross-linking. The second system used BMEM raised against and specific for the hapten 4-hydroxy-3-nitrophenylacetyl (NP), with this system having been previously characterized (McHeyzer-Williams et al., 1991; Lalor et al., 1992). The third system used BMEM from a transgenic mouse in which the polyclonal BMEM population is readily identified. This latter source allowed for the isolation of BMEM without the need for BCR engagement by antigen, as when PE-BMEM and NP-BMEM are purified using antigen binding to denote specificity, cross-linking the BCR is inevitable.

PE-BMEM cells.

From PE-immunized mice, PE-BMEM were first enriched by negative selection, and then electronically sorted based on a B220+PE+dumpCD38+ phenotype, with post-sort analysis indicating high levels of purity (Fig. S2 A).

NP-specific BMEM cells.

NP-BMEM were generated in a transgenic mouse strain where Cre recombinase was inserted in the Cγ1 locus, with this strain crossed with the Rosa26-EYFP reporter mouse (Casola et al., 2006). In this mouse, every post-GC B cell expresses YFP, with this strain affording the opportunity to track post-GC B cell subsets by YFP expression (Casola et al., 2006). Immunization of Cγ1-cre/Rosa26-EYFP mice with NP-keyhole limpet hemocyanin (KLH)/alum lead to the generation of NP-BMEM (Fig. S2 B; Casola et al., 2006). After negative enrichment, NP-BMEM were defined as B220+ NIP+IgG1/YFP+dumpCD38+, with post-sort analysis indicating purity (Fig. S2 B).

Polyclonal BMEM cells.

Polyclonal BMEM can be clearly enumerated in Cγ1-cre/Rosa26-EYFP mice through expression of YFP, loss of IgD and IgM, and expression of other BMEM markers. NP-immunized Cγ1-cre/Rosa26-EYFP mice were negatively enriched for BMEM with polyclonal BMEM defined as B220+IgG1/YFP+CD38+dumpCD80+ and sorted to purity (Fig. 1 C).

NF B cells.

In parallel, NF B cells were defined as B220+CD23+IgD+CD38+ and were additionally sorted (Fig. S2 C).

The response of PE-BMEM, NP-BMEM, poly-BMEM, and NF B cells to TLR agonists after 3 d of in vitro culture was analyzed (Fig. 1). PE-BMEM and corresponding NF B cells and IL-4–only controls were stained with CFSE, whereas NP-BMEM and poly-BMEM with NF B cells and IL-4 only controls were stained with DDAO to track proliferation. After culture with the TLR4 agonist LPS, cells were analyzed for surface expression of B220 and CD138. The culture of all NF and BMEM with IL-4 resulted in a uniform B220+ population after 3 d (unpublished data). Upon culture of NF B cells and BMEM with LPS, NF B cells remained primarily B220+CD138, whereas all three sources of BMEM generated a B220intCD138+ population of cells (∼21–30% of total) representing the phenotype of a ASC (Fig. 1, A and D). The ASCs derived from the three sources of BMEM were of the IgG isotype and, with the ASCs generated from PE-BMEM and NP-BMEM, specific for either PE or NP, respectively (Fig. 1 G and not depicted). NF B cells generated a high frequency of IgM+ ASCs (Fig. 1 G). The overall degree of proliferation by the three sources of BMEM was moderately less than NF B cells, although proliferation did occur, as indicated by comparison to the IL-4–only cultured cells (Fig. 1, B and E). The population of B220intCD138+ cells generated from poly-BMEM arose in the daughter peaks, showing that BMEM undergo cell division while differentiating into B220intCD138+ cells (Fig. 1 F).

The response of poly-BMEM and NF B cells was analyzed to either Pam3CSK4 (TLR1/2 agonist), heat-killed Listeria monocytogenes (TLR2 agonist), poly(I:C) (TLR3 agonist), LPS (TLR4 agonist), flagellin from Salmonella typhimurium (TLR5 agonist), FSL-1 (TLR6 agonist), S-27609 (TLR7 agonist), ODN 1826 (TLR9 agonist), and IL-4. Both NF B cells and poly-BMEM were responsive to agonists for TLR1/2, TLR4, TLR6, TLR7, and TLR9 (Fig. 1 I). Both poly-BMEM and NF B responded poorly or did not respond to agonists for TLR2, TLR3, and TLR5 (unpublished data). For each TLR agonist that NF B cells or poly-BMEM responded to, BMEM generated a sizable population of B220intCD138+ cells, whereas NF B cells maintained a B220+CD138 phenotype (Fig. 1 H). NF B cells also proliferated to a greater degree than BMEM (Fig. 1 I). Finally, it was observed that while engagement of CD40 drove proliferation of both BMEM and NF B cells, BMEM did not differentiate into B220intCD138+ cells (Fig. 1, H–I). Collectively, these data show that BMEM are sensitive to multiple TLR agonists in vitro and do not require BCR cross-linking to become activated and differentiate into ASCs. Furthermore, upon activation, BMEM have an enhanced intrinsic capacity to differentiate into CD138+ ASCs compared with NF B cells. Lastly, CD40 signaling appears to drive BMEM proliferation, but to block differentiation in vitro.

BMEM cells undergo rapid clonal expansion and differentiation into IgG+ ASCs in response to soluble antigen

Having established that BMEM are responsive to bystander stimuli in vitro, we used the PE-antigen system to compare BMEM responsiveness toward cognate antigen versus bystander inflammation in vivo. We first performed a detailed analysis of the secondary humoral immune response to soluble PE (sPE) to define the behavior and kinetics of BMEM activation in vivo. Unlike naive hosts, it is well-established that previously immunized mice are responsive to low-doses of antigen in the absence of adjuvant (McHeyzer-Williams et al., 2006). This heightened responsiveness results from the combined presence of antigen-reactive BMEM and CD4+ memory T cells. In concordance with this, we observed that injection of naive mice with 1 µg sPE (or 10 µg; not depicted) failed to elicit PE-ASCs (Fig. 2, A and B), whereas PE-immunized mice produced high numbers of IgG+ PE-ASCs. A robust IgM ASC response was only elicited when naive or PE-immunized mice were injected with 1 µg of sPE along with the TLR9 agonist CpG ODN 1826 (CpG; Fig. 2 A). Thus, the IgG+ PE-specific humoral response generated after sPE injection is likely the direct consequence of BMEM activation. These data demonstrate that a low level of sPE given in the absence of adjuvant is capable of activating PE-BMEM to differentiate into IgG+ ASCs.

The in vivo proliferation of PE-BMEM was measured by flow cytofluorimetric analysis of BrdU incorporation. PE-immunized mice were injected with sPE, and a 5-d BrdU pulse was performed starting on the day of sPE injection, followed by a chase period. BrdU incorporation by BMEM was quantified at various times during and after the pulse period. After preenrichment, PE-BMEM were detected by excluding CD4/CD8/IgD/IgM+ cells via a dump gate and gating them on B220+PE+ cells, with these cells analyzed for BrdU incorporation (Fig. S3 A). PE-immunized mice receiving BrdU, but no antigen, and immunized mice receiving neither antigen nor BrdU were included as negative controls (Fig. 2 C). 2 d after sPE rechallenge, BMEM began to proliferate, with this response peaking by day 4 (Fig. 2 C and Fig. S3 A). In mice analyzed during the BrdU chase period (days 11 and 24 after sPE rechallenge), a proportion of BMEM remained BrdU+ at a percentage similar to that observed at the peak of the response (35.5% ± SEM 4.6 and 38.4% ± SEM 5.1 versus 48.9% ± SEM 5.8, respectively), indicating that 71–77% of BMEM proliferation occurs within the first 5 d after antigen rechallenge (Fig. 2 C and Fig. S3 A). A second PE-binding population was also observed in these experiments, with this population displaying a B220PE+ phenotype (Fig. S3 A). When the BrdU incorporation frequencies by the B220PE+ population in PBS versus sPE-immunized mice were compared, no differences were observed between the two groups over the 24-d kinetic analysis (unpublished data). This indicates that the B220PE+ population does not respond to sPE, and as antigen-responsiveness is a crucial element of adaptive memory, we excluded these cells from further analysis. As BrdU labeling measures the accumulation of proliferation over the time of the BrdU “pulse” period, we also analyzed BMEM expression of Ki-67 at various times after antigen challenge (Fig. 2 F). Ki-67 expression is an indicator of all stages of the cell cycle and provides a snapshot of the proliferative state of the BMEM at a given point in time (Gerdes et al., 1984). PE-BMEM are almost uniformly Ki-67, which is indicative of their quiescent state (Fig. 2 F). By days 3 and 4 after sPE rechallenge, 76–77% of BMEM cells were Ki-67+. At day 5, Ki-67 expression began to dissipate, and most PE-BMEM exhibited decreased Ki-67 expression on day 7, with this decrease more pronounced by day 11. By day 24, PE-BMEM were once again uniform in their lack of Ki-67 expression (Fig. 2 F). In correlating the expression pattern of Ki-67 by BMEM (Fig. 2 F) with the 5-d BrdU pulse followed by a chase period (Fig. 2 C), the results indicate that the majority of BMEM proliferation occurs between days 2 and 5 after sPE rechallenge. In addition, the presence of BrdU+ BMEM 24 d after sPE injection provides direct evidence that BMEM generate daughter BMEM after secondary antigen encounter.

A rapid increase in the numbers of IgG+ PE-ASCs in both the spleen and BM was observed 3 d after sPE rechallenge (Fig. 2, B and D). This burst in ASC numbers peaked 4 d after challenge in the spleen and 5 d in the BM, after which ASC numbers gradually subsided over the next 20 d (Fig. 2 D). Corresponding to the increase of ASCs after rechallenge was an increase in anti-PE IgG serum titers increasing 10-fold over the next 20 d (Fig. 2 E). These data show that ASCs are rapidly generated within 3 d after antigen rechallenge and are present to substantial degrees in both the spleen and the BM. Soon thereafter, ASC numbers declined, indicating that short-lived ASCs are present in both organs.

The observation that ∼50% of BMEM respond to sPE as indicated by BrdU incorporation (Fig. 2 C and Fig. S3 A) led us to question whether either the antigen dose or the absence of adjuvant during antigen administration was limiting the frequency of responding BMEM. To test this, we varied the amount of sPE injected or added CpG during antigen rechallenge, with BrdU administration concurrently initiated. When analyzed 3 d later, it was observed that the dose of antigen positively correlated with the percentage of proliferating BMEM (Fig. 2 G and Fig. S3 B). The addition of CpG had no further impact on the frequency of responding PE-BMEM (Fig. 2 G and Fig. S3 C). However, the addition of CpG to sPE modestly increased the number of splenic IgG+ PE-ASCs elicited after 3 d when compared with sPE alone (Fig. 2 B). In sum, these data indicate that antigen can be a limiting factor in dictating the frequency of responding BMEM. Notably, TLR9 agonists failed to enhance the frequency of activated BMEM. This point indicates that CpGs alone may not play a role in driving in vivo BMEM activation.

BMEM cells proliferate upon in vivo antigen encounter and do not express GC markers or generate GC structures

The rapid kinetics of the BMEM response led us to question whether BMEM form GCs after antigen encounter. Clonally expanding BMEM were tracked to define their transitional stages en route to differentiating into ASCs. Under steady-state conditions, PE-BMEM displayed a GL7PNAKi-67CD38+ phenotype as anticipated (Fig. 3 A; Lalor et al., 1992). As a positive control for GC markers, Peyer's patches were harvested and gated on B220+IgD cells, a population known to be primarily GC B cells, with these cells exhibiting a GL7+PNA+Ki-67hiCD38 phenotype (Butcher et al., 1982). When PE-BMEM were harvested and analyzed 3 d after sPE challenge, they were GL7PNAKi-67hiCD38+, indicating these cells had responded to antigen because of Ki-67 expression, yet failed to express the GC markers GL7 and PNA (Fig. 3 A). It was plausible that the failure of antigen-responding BMEM to gain a GC phenotype was caused by the solitary use of sPE, and that the induction of a GC phenotype requires the presence of an adjuvant. Therefore, CpG was added to sPE, and 3 d later, the phenotype of the responding BMEM was analyzed. The addition of CpG to sPE again elicited GL7PNAKi-67hiCD38+ BMEM (Fig. 3 A). It was considered possible that BMEM gain a GC phenotype later than 3 d after antigen encounter, so the phenotype of BMEM was analyzed at later points in time. The results indicate that PE-BMEM maintain a GL7PNACD38+ phenotype throughout the cell cycle (Fig. 3 B). These data define a proliferating state of BMEM occurring shortly after in vivo antigen encounter, with these BMEM expressing a GL7PNAKi-67hiCD38+ phenotype.

To investigate whether antigen-activated BMEM generate GCs, GC structures were quantified in spleens where PE-BMEM were either unchallenged or were responding to sPE or sPE + CpG by immunofluorescence (IF) microscopy. As a positive control for the presence of GCs, naive BALB/c were immunized with PE/alum and spleens were examined 10 d later at the height of the GC response. To ensure that PE-BMEM in the sPE- and sPE + CpG–treated groups and the PE-GC B cells in the D10 PE/alum immunized groups were present and proliferating in these mice, FACS analysis was performed on half of the spleens, with BrdU incorporation by B220+PE+ cells analyzed (Fig. S4). Upon IF evaluation, in the D10 PE/alum immunized spleens, GL7+ clusters were observed and proximal to CD35+ follicular DCs, with these structures detected in ∼50% of the B cell follicles (Fig. 3, C and D). These GL7+ cell clusters were minimally present in the PBS-, sPE-, and sPE + CpG–treated groups (Fig. 3, C and D). Importantly, there was no significant increase in the number of GCs present in spleens where BMEM were activated by sPE or sPE + CpG in comparison to PBS controls (Fig. 3 D). These data, coupled with the GL7PNAKi-67hiCD38+ phenotype displayed by BMEM after in vivo antigen-dependent activation, demonstrate that GCs are not 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, combinations of TLR agonists, agonistic αCD40, and/or αCD3. 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 (unpublished 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 proliferation 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 proliferate in response to injection with agonistic αCD40 antibody alone or coupled with LPS or CpG, with the mean proliferation of both subsets as measured by BrdU incorporation between 25 and 50% (Fig. 4, A and B, Fig. S5). After injection with LPS, CpG, or αCD3 alone or αCD3 and LPS or CpG, no proliferation was observed for either NF B cells or PE-BMEM (Fig. 4, A and B, and Fig. S5). These data indicate that both NF B cells and PE-BMEM proliferate in response to αCD40 signals in vivo, whereas TLR agonists or activation of T cells with αCD3 did not induce BMEM proliferation.

To determine whether BMEM differentiate into ASCs after signaling through CD40, PE-immunized mice were injected with PBS, sPE, CpG, αCD40, or both αCD40 and CpG. IgG+ PE-ASCs after agonist injection were quantified, and only in the sPE challenged mice was an increase in IgG+ PE-ASC numbers observed (unpublished data). The numbers of BM IgG+ PE-ASCs after agonist treatment were also analyzed, with serum harvested on the day of agonist injection and again when analyzed 14 d later. The only treatment group in which an increase in numbers of BM IgG+ PE-ASCs and serum PE-specific IgG was observed in the sPE-treated cohort (Fig. 4, C and D).

These data indicate that TLR4 and TLR9 agonists and the proinflammatory cytokine storm elicited by agonistic αCD3 treatment are incapable of activating BMEM and driving their proliferation or differentiation in vivo. Engagement of CD40 (either alone or coupled with TLR4 and TLR9 agonists) drives both NF B cell and BMEM proliferation, but is incapable of mediating their differentiation into ASCs.

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 administered with CpG or infection with vaccinia virus Western 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 immunization 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 with our earlier observations (Fig. 2, D and E), sPE induced a substantial and significant increase in the number of IgG+ PE-ASCs in both the spleen and BM (Fig. 5, B and C) and led to an ∼30-fold increase in the levels of IgG+ PE-specific serum antibody titers (Fig. 5 D). Both cellular and humoral anti-VACVWR immune responses were verified to have occurred by testing for the presence of IFN-γ+ VACVWR-specific CD4 and CD8 T cells in spleens harvested from VACVWR-infected mice, but not naive mice, and by observing for the presence of anti-VACVWR IgG serum antibodies only in infected mice (Fig. S6, A and B).

These data collectively demonstrate that immunization with NP-KLH and VACVWR infection does not induce PE-BMEM proliferation or differentiation into ASCs. Thus, BMEM are unresponsive to stimuli generated during a bystander immune response.

DISCUSSION

A detailed analysis of the in vivo secondary immune response of BMEM in response to cognate antigen versus noncognate inflammatory bystander signals is presented. These data show that BMEM respond robustly upon rechallenge with cognate antigen, but do not acquire the expression of GC markers or form GCs in response to cognate antigen. In striking contrast, the response of BMEM to a battery of proinflammatory signals is remarkably silent. It has been proposed that serological memory is sustained by BMEM responding to bystander inflammatory signals by undergoing proliferation and differentiation. However, the findings of this study indicate that BMEM ignore these signals (TLR agonists, polyclonal T cell activation, aggressive vaccination, and acute viral infection), as they fail to drive BMEM proliferation or differentiation in vivo.

Despite the preponderance of studies detailing the B cell dynamics of the primary humoral immune response, surprisingly few studies have detailed the events of the secondary humoral immune response. We have tracked BMEM and demonstrate that cognate antigen drives the rapid clonal expansion of BMEM and their terminal differentiation into ASCs, with these events occurring in the absence of GC formation. As has been widely reported, BMEM rapidly differentiate to ASCs upon secondary antigen encounter (Höfer et al., 2006; Minges Wols et al., 2007), with this ASC response first detectable 3 d after antigen challenge in both the spleen and the BM and with the splenic ASC response an order of magnitude greater than that observed in the BM. This wave of ASCs dissipates after 11 d, and it is probable that the remaining ASCs in the BM 24 d after antigen challenge have been culled into the long-lived PC pool (Manz et al., 1997; Höfer et al., 2006; Minges Wols et al., 2007). These data detail the activation of BMEM and define their proliferation as occurring between days 2 and 5 after antigen encounter. Proliferating BMEM occupy a transitional state defined as antigen-binding B220+CD38+Ki-67+GL7PNA before generating quiescent daughter BMEM and/or differentiating into ASCs. This blasting state is unique because of the presence of high levels of CD38 expression by proliferating cells; CD38 is a phenotypic marker previously used to denote quiescent NF B cells and BMEM and is lost upon B cell activation and entry into the GC (Oliver et al., 1997). Our finding that BMEM cells do not form or enter a GC reaction during their differentiation and expansion is consistent with classical studies indicating that BMEM might not undergo further SHM and isotype switching upon rechallenge with antigen (Askonas et al., 1970; Okumura et al., 1976; Siekevitz et al., 1987). In these studies, limiting numbers of hapten-specific “parental” BMEM were adoptively transferred together with carrier-primed T cells into lightly irradiated hosts, followed by immunization of the hosts with soluble hapten-carrier antigen. Upon analyzing the isotype distribution and SHM exhibited by the parental BMEM and their progeny generated in response to cognate antigen, no change in diversity in either measure was observed. Our data showing that BMEM neither gain GC markers nor seed GCs are in concordance with these studies. It is apparent that BMEM, at least in these studies and in our presented data, are not instructed to enhance the affinity or isotype distribution of their antigen receptors during secondary antigen encounter.

Our data indicate that quiescent IgG+ BMEM are not activated by TLR agonists or bystander T cell help, and thus do not undergo self-renewal events or differentiate into ASCs in an antigen-independent manner. We should note that our analysis was limited to IgG+ PE-BMEM and that our experimental system excluded testing whether IgM+ BMEM are responsive to bystander inflammatory signals. There is a growing body of evidence both in favor of and in conflict with the hypothesis that serological memory is sustained by the activation of BMEM by bystander inflammation. Evidence in favor of this theory includes in vitro observations in which human BMEM were shown to have increased sensitivity and to selectively proliferate and differentiate into ASCs in response to bystander stimuli as compared with NF B cells (Bernasconi et al., 2002, 2003). In vivo studies in humans found that for single-pathogen specificities, serum antibody levels linearly correlated with the frequency of BMEM present in a host, indicating that the ASC compartment was genealogically linked to and undergoing replenishment by the BMEM compartment (Bernasconi et al., 2002). Mouse studies also supported this link between the BMEM and ASC compartments as elimination of virus-specific BMEM led to a slow decay of virus-specific ASC numbers over time in comparison to mice still containing BMEM (Slifka et al., 1998). Lastly, it was observed that during an immune response against a single pathogen, an increase in both blood ASC numbers and serum antibody levels occurred for specificities to other pathogens (Bernasconi et al., 2002). This result was interpreted as evidence that host BMEM were recipients of antigen-independent stimuli and responded by differentiating into ASCs.

Despite these data suggesting that BMEM respond to bystander inflammation, more recent studies have put this hypothesis under scrutiny. In particular, further attempts to correlate antigen-specific BMEM numbers with circulating ASCs in human subjects have been unsuccessful (Amanna et al., 2007). Mouse studies where long-lived ASC numbers were quantified after BMEM depletion observed no decay in ASC numbers over time, contrary to initial reports (Ahuja et al., 2008; DiLillo et al., 2008). Lastly, a series of studies reported that in mice that were administered a TLR agonist or in humans that were given a vaccine, ASC numbers and serum antibody levels against specificities other than the immunizing antigen did not increase (Di Genova et al., 2006; Amanna et al., 2007; Xiang et al., 2007; Richard et al., 2008).

Our studies show that the in vitro sensitivities of BMEM to activation are not representative of their sensitivities in vivo and cast doubt on the hypothesis that BMEM can be activated by bystander inflammation. Our in vitro studies examining the response profiles of murine BMEM to both antigen-dependent and -independent stimuli extended prior studies using human (Arpin et al., 1997; Bernasconi et al., 2002) and murine (Richard et al., 2008) BMEM, as we observe that murine BMEM are sensitive to TLR agonists and have an enhanced intrinsic ability to differentiate into ASCs upon activation with TLR agonists, regardless of whether the BCR is engaged by antigen. Given these data, and our knowledge of the robust response of BMEM to cognate antigen in vivo, an exploration of the response of BMEM to several nonspecific inflammatory signals in vivo demonstrated that BMEM are in fact unresponsive to these stimuli. The first panel of signals tested was derived from activated T cells and included CD154 expression by these cells, as well as proinflammatory cytokines either alone or together with TLR agonists. We first analyzed the impact of agonistic αCD40 antibodies on BMEM proliferation and differentiation. The injection of mice with αCD40 alone or with TLR4 or TLR9 agonists elicited substantial 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 αCD40 induced the proliferation of, yet failed to differentiate, poly-BMEM into ASCs. These studies also extend previous in vitro studies where CD40 signaling triggered human 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 αCD3 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 release a proinflammatory cytokine storm that includes IL-2, IFN-γ, 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 antigen 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 signaling drives BMEM proliferation but blocks their differentiation into ASCs, indicating that differentiation occurs upon cessation 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 activation, and adaptive immune responses are incapable of driving 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 populations not requiring self-renewal or replenishment and with serological memory not requiring maintenance. However, this hypothesis requires that BMEM and PCs survive for perpetuity, and this quality appears unrealistic. From our presented work, we show that BMEM do not clonally expand or differentiate 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 process, with BMEM not requiring an extrinsic signal to maintain serological memory such as those tested herein.

MATERIALS AND METHODS

Mice and immunizations.

These studies were approved by the Institutional 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. Cγ1-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 infections, 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 treatment, 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 of BMEM 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 Enrichment Kit.

Cell culture/reagents.

B cells were cultured in RPMI media supplemented with 10% FBS (Atlanta Biologicals), Hepes, 50 µM β-ME, and penicillin/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 × 106 splenocytes harvested 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. Intracellular staining of IFN-γ 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 phosphorothioate 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), αCD40 clone FGK45, and αCD3 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-γ (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 manufacturers 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).

ELISpot/ELISA analysis.

For ELISpot analysis, cells were apportioned to PE, NP3-BSA, or polyclonal anti-Ig–coated Multiscreen 96-well plates (Millipore) with twofold serial dilutions made before incubation for 7–18 h at 37°C. PCs were detected by HRP-conjugated or biotinylated anti–mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgE, or IgA polyclonal antibodies (SouthernBiotech.), with streptavidin-HRP used in a second step when necessary. ELISpots were developed as previously described (Benson et al., 2008).

For ELISA analysis, plates were coated with either 10 µg/ml PE or 4 × 106 PFU of H2O2-inactivated VACVWR (Hammarlund et al., 2008) overnight in PBS, blocked with PBS + 5% FBS, and washed, and then 1:2,000 diluted serum added at serial 1:2 dilutions. Serum from either a PE hyperimmunized mouse or from a VACVWR mouse was included on each plate as a reference between plates and between experiments and used to generate a standard curve, with these serum allotted the value 8,000 arbitrary units. Antibody levels were detected with IgG-AP (SouthernBiotech) and developed with 1 mg/ml pNPP (Sigma-Aldrich) in 0.05 sodium carbonate buffer.

Immunofluoresence microscopy.

Spleens were presoaked in 15% sucrose/PBS for 30 min and snap-frozen in OCT compound (Sakura Finetek) in a dry ice/ethanol slurry and stored at −80°C. Tissues were sectioned onto slides using a cryostat with 8-µm-thick sections. After drying at room temperature, the tissue was fixed in ice-cold 1:1 acetone/methanol mix for 10 min and allowed to dry, after which sections were outlined with an ImmEdge Pen (Vector Laboratories). Sections were blocked with 10% heat-inactivated normal rat serum/PBS for 1 h, washed, and stained with IgD-Alexa Fluor 647, GL7-FITC, and CD35-biotin, followed by staining with SA-PerCP. After washing, slides were mounted with PermaFluor Aqueous Mounting Media (Thermo Fisher Scientific). Samples were analyzed on a LSM 510 META confocal microscope using LSM software (Carl Zeiss, Inc.).

Online supplemental material.

Fig. S1 shows FACS gating scheme and phenotype of PE-BMEM. Fig. S2 displays the FACS profiles and gating schemes used to sort-purify PE-BMEM, NP-BMEM, and NF B cells. Fig. S3 contains FACS profiles showing BrdU incorporation by PE-BMEM and data corresponding to experiments presented in Fig. 2. Fig. S4 contains FACS plots showing BrdU incorporation by PE-BMEM and D10 GC B cells, with these data corresponding to experiments presented in Fig. 3. Fig. S5 contains FACS plots showing BrdU incorporation by PE-BMEM and NF B cells after in vivo administration of TLR agonists, CD40 agonists, and bystander T cell help. Data correspond to experiments presented in Fig. 4. Fig. S6 depicts the presence of VACVWR-specific CD4 and CD8 cells and VACVWR–specific serum IgG in infected hosts. FACS plots are also depicted showing levels of BrdU incorporation by PE-BMEM after protein vaccination or viral infection, with data corresponding to experiments depicted in Fig. 5.

Acknowledgments

We thank Dr. Klaus Rajewsky and Dr. Stefano Casola for providing the Cγ1-cre mice. We thank Dr. Stacey Dillon and ZymoGenetics, Inc. (Seattle, WA) for providing some of the reagents used in these studies.

This work was supported by National Institutes of Health (NIH) grants RO1A1026296 and RO1A108896 to R.J. Noelle and NIH pre-doctoral training grant T32AI07363 to M.J. Benson.

The authors have no conflicting financial interests.

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Abbreviations used: ASC, antibody-secreting cell; BCR, B cell receptor; BMEM, memory B cell; GC, germinal center; IF, immunofluorescence; KLH, keyhole limpet hemocyanin; MZ, marginal zone; NF, naive follicular; NP, 4-hydroxy-3-nitrophenylacetyl; PB, plasmablast; PC, plasma cell; PE, phycoerythrin; SHM, somatically hypermutated; sPE, soluble PE; TLR, Toll-like receptor; VACVWR, vaccinia virus Western reserve strain.

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