Cellular dynamics of memory B cell populations: IgM⁺ and IgG⁺ memory B cells persist indefinitely as quiescent cells

Abstract

Despite their critical role in long-term immunity, the lifespan of individual memory B cells remains poorly defined. Using a tetracycline-regulated pulse-chase system, we measured population turnover rates and individual half-lives of pre-established antigen-induced immunoglobulin (Ig) class-switched and IgM-positive memory B cells over 402 days. Our results indicate that, once established, both IgG-positive and less frequent IgM-positive memory populations are exceptionally stable, with little evidence of attrition or cellular turnover. Indeed, the vast majority of cells in both pools exhibited half-lives that appear to exceed the lifespan of the mouse, contrasting dramatically with mature naïve B cells. These results indicate that recall antibody responses are mediated by stable pools of extremely long-lived cells, and suggest that antigen-experienced B cells employ remarkably efficient survival mechanisms.

Introduction

Memory B cells mediate robust recall responses by differentiating rapidly into antibody-secreting plasma cells and forming nascent germinal centers (GCs), and by serving as effective antigen presenting cells (1–4). Because secondary antibody responses can be induced long after initial antigen encounter (5), it is generally assumed that individual memory B cells are exceptionally long-lived. However, current experimental evidence for this idea is varied and at times contradictory. Yet, a clear definition of memory B cell lifespans and the mechanisms regulating this process is critical for vaccine design and for developing improved strategies for combating antibody-mediated pathologies.

In people numbers of antigen-specific memory B cells remain relatively stable for more than 50 years after smallpox vaccination (6). However these results do not provide information on the lifespan of individual cells. Consequently, it is not known whether long-term maintenance of such populations requires periodic input from activated B cells, or whether particular clones come to dominate memory pools over extended time frames. The former scenario is consistent with the work of Barrington et al. wherein persisting antigen appeared to promote the generation of nascent memory B cells well after immunization (7). Similarly, others have proposed that maintenance of serum antibody titers requires the slow but consistent generation of plasma cells by antigen-activated memory B cells (8, 9). These ideas are consistent with an a model put forth earlier by Fearon and colleagues proposing that memory B cells employ a stem cell-like self renewal program to continuously generate plasma cells (10). Notably, although each of these scenarios predicts that memory B cell pools contain meaningful numbers of activated or recently activated cells, there is little information on the steady-state dynamics of established memory B cell populations.

Several groups have since sought to define the lifespan of memory B cells, however the results have not led to a clear consensus. Using a B cell receptor (BCR) transgenic system, Anderson et al. showed that memory B cell numbers remained constant between 8–20 weeks post-immunization, and based on short-term in vivo BrdU labeling experiments estimated the half-life of memory B cells to be 8–10 weeks (11). Given that the accepted half-life of naïve B cells is 7–8 weeks (12–14), based on these results it is unclear whether individual memory B cells possess substantially longer lifespans than their naïve counterparts. By contrast, Pape et al. showed immunization of conventional inbred mice with the protein phycoerythrin (PE) induced the generation of long-lived IgM⁺ and class-switched (IgG⁺) memory cells (15). However, whereas in this system IgM⁺ memory B cells remained constant for upwards of 500 days, class-switched cells decayed with exponential kinetics, returning to pre-immunization levels by 400 days (15). Why IgM and class-switched memory cells might possess distinct half-lives remains to be determined. These results also appear to differ with those of Schittek and Rajewsky, who showed that class-switched memory B cell pools are relatively stable over 8 weeks (16). However, the latter workers did not examine decay rates for extended periods, or attempt to calculate half-lives for individual cells within this pool.

To resolve these issues we employed a non-toxic pulse-chase labeling approach. This strategy exploits a tetracycline-regulated reporter allele encoding the chromatin protein histone 2B fused to GFP. This approach allowed us to establish decay rates for individual cells within established antigen-specific memory B cells populations without concern for the toxic effects associated with extended exposure to DNA nucleotide analogs such as BrdU. To provide appropriate benchmarks for this system we also determined decay rates for naïve B cell populations. Our results show that whereas naïve follicular and marginal zone B cells exhibit decay rates consistent with a half-life of 13–22 weeks, decay rates for IgM⁺ and IgG⁺ memory B cells were markedly slower, revealing cellular half-lives greater than the 2-year lifespan of the mouse. These data illustrate that, once established, antigen-specific memory B cell populations are remarkably stable and highly enriched for quiescent and exceptionally long-lived cells.

Materials and Methods

Mice and immunizations

Adult C57BL/6 or C57BL/6-backcrossed Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mice were purchased from Jackson Laboratories and maintained in a specific pathogen-free facility at the University of Pennsylvania, in accordance with institutional guidelines for animal care and welfare. Mice were immunized i.p. with 50µg NP₁₈-chicken γ-globulin (CγG) in alum. To induce transgene expression, mice were maintained on drinking water containing 2mg/mL doxycycline (Sigma) supplemented with 10mg/mL sucrose.

Flow cytometry

Single-cell suspensions of splenocytes were prepared, depleted of red blood cells by hypotonic lysis, and stained with optimal dilutions of the indicated antibodies, as described (17). All of the following reagents were obtained from eBioscience, unless noted otherwise: PerCP-eFluor710-anti-IgM (II/41); PE anti-CD23 (B3B4; BD Pharmingen), anti-CD138 (281-2; BD Pharmingen); PE-Texas Red anti-B220 (RA3-6B2); PE-Cy7 anti-CD21/35 (eBioBD9), anti-CD4 (GK1.5), anti-CD8α (53-6.7), anti-Gr-1 (RB6-8C5), anti-F4/80 (BM8), and anti-TER119 (BioLegend); allophycocyanin anti-CD93 (AA4.1); AlexaFluor700 anti-CD38 (90); allophycocyanin-Cy5.5 anti-CD19 (RM7719; Invitrogen); allophycocyanin-Cy7 anti-IgD (11-26c.2a; BioLegend); Brilliant Violet (BV; all from BioLegend) 605 anti-CD73 (TY/11.8), BV650 anti-CD80 (16-I0A1), BV785 anti-CD19 (6D5), and biotin-anti-PDL2 (TY25; BioLegend). Biotinylated antibodies were revealed with streptavidin-BV421 (BioLegend). NP-allophycocyanin was conjugated in-house, using standard methods. Dead cells were excluded from analyses using Zombie Aqua (BioLegend), and doublets were excluded using the combined width parameter of the forward and side scatter parameters. Antibodies for B cell-activating factor belonging to the TNF family (BAFF)-receptor (BAFF-R) and transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) were PE conjugates purchased from BioLegend. Flow cytometric acquisition was performed on a BD LSRII, and analyses were performed using FlowJo 8.8.6 (Tree Star). Multiple files per sample were concatenated prior to data analysis. Cells were sorted for cell culture with a 4-laser FACSAria.

Cell culture

Sorted B cells were cultured for 2–3 days in RPMI-1640 supplemented with 10% FCS and glutamine, antibiotics, HEPES, and 2-ME before analysis by flow cytometry. LPS (E. coli) was purchased from Sigma and used at 10 µg/ml.

Estimation of cellular half-life

When possible half-lives for defined cell populations were calculated as follows: t½ = (T × log2)/log (starting quantity/ending quantity), where T = elapsed time.

Results

Inclusion of the tetracycline analog doxycycline (DOX) in the drinking water of Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mice induces the expression of Histone2B-GFP fusion proteins. Once generated, Histone2B-GFP proteins incorporate into the chromatin of most cells, allowing the tracking of individual non-dividing cells for at least one year after terminating DOX exposure (18). We immunized adult B6-backcrossed Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mice with immunogenic conjugates of (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to chicken γ-globulin (CγG). This approach allows direct identification of NP-specific B cell populations by flow cytometry using NP conjugates of the fluorescent protein allophycocyanin (APC) (19). Because limiting cell extrinsic factors may be needed for memory B cell generation and survival, we did not employ Ig transgenic mice to increase frequencies of responding cells. To characterize antigen-experienced B cells, we focused on NP-binding CD19⁺ IgD⁻ cells that also lacked the non-B cell surface antigens (“Dump”) CD4, CD8α, Gr-1, F4/80 and TER-119 (19). We also focused on B220⁺ CD38⁺ cells to avoid persisting GC B cells (2). With this strategy we were able to detect NP-specific B cells for at least 402 days post-immunization (Fig. 1A, C). The vast majority of NP-binding CD38⁺ CD19⁺ B220⁺ (Dump⁻) B cells uniformly expressed the memory B cell surface phenotype CD73⁺ PD-L2⁺ at every time point (Fig. 1A, B, not shown) (20). Consistent with several reports showing that Igλ⁺ B cells dominate the NP-specific antibody response in B6 mice (21–23), the majority of NP-binding cells were Igλ⁺; yet it should be emphasized that some 30% of these cells were Igκ⁺ (Fig. 1B).

Figure 1. Long-term resolution of hapten-specific memory B cells.

(A) Splenocytes from an adult B6.Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mouse immunized with NP-CγG 402 days previously were stained with the indicated antibodies and NP-APC to detect NP-binding B cells. The left-most plot was pre-gated for viable lymphocytes. Representative of 3–5 mice per time point. The right-most plot illustrates background for NP-binding B cells in a mouse that was not immunized. (B) Expression of the indicated surface molecules on populations gated in (A). (C) Absolute numbers per spleen of NP-binding IgD⁻ Dump⁻ CD19⁺ B220⁺ CD38⁺ cells at each time point. Each circle represents a single mouse; means and standard deviations for each time point are indicated by hash marks and error bars, respectively. The limit of detection represents the means plus 2 SEMs for NP-binding B cells from seven non-immunized controls. For each sample at least 10 × 10⁶ events/file were collected on an LSR2 flow cytometer.

Notably, absolute numbers of NP-binding CD38⁺ CD19⁺ B220⁺ cells remained constant between 86 and 402 days post-immunization (Fig. 1C). Furthermore, antigen-induced NP-binding B cells defined by these criteria could be readily subdivided into either IgM⁺ or IgG⁺ subpopulations, and both fractions were highly enriched for CD73⁺ PD-L2⁺ cells (Fig. 2A) Therefore this strategy allowed us to identify both IgM⁺ and class-switched memory B cells. Consistent with this conclusion, as shown previously for bulk memory B cells in mice and humans (24, 25), both IgM⁺ and IgG⁺ antigen-induced NP-binding cells expressed increased surface levels of the BAFF-receptor TACI, whereas levels of the primary BAFF receptor BAFF-R (or BR-3) were similar on naïve and all antigen-experienced B cells examined (Fig. 2B). Moreover, consistent with the notion that memory B cells generate plasma cells with increased kinetics compared to naïve B cells (26, 27), when stimulated with LPS NP-binding IgD⁻ CD38⁺ CD19⁺ cells generated CD138⁺ plasma cells faster and more effectively than naïve follicular B cells (Fig. 2C). Altogether, these results show that NP-specific memory B cells achieve steady state within 86 days post-immunization and can be detected for extended periods post-immunization without employing Ig transgenic mice.

Figure 2. Characteristics of IgM⁺ and IgG⁺ NP-specific memory B cells.

(A) Splenocytes from a C57BL/6 adult immunized with NP-CγG 54 days previously were stained with the indicated reagents. The left-most plot was pre-gated as shown in Figure 1A. 10 × 10⁶ events were collected on an LSR2 flow cytometer. (B) Splenocytes from a C57BL/6 mouse immunized with NP-CγG 117 days previously were stained as shown in the left-most plot in (A) and with either anti-BAFF-R or anti-TACI antibodies. 10 × 10⁶ events were analyzed. Background staining (filled gray histogram) was established by gating on Dump⁺ cells (not shown). (C) NP-binding IgD⁻ Dump⁻ CD19⁺ B220⁺ CD38⁺ cells harvested from C57BL/6 mice 117 days post-immunization, or naïve follicular (CD19⁺ CD23⁺ CD21^int. AA4⁻) or marginal zone (CD19⁺ CD23⁻ CD21^high AA4⁻) B cells sorted from naïve adults were stimulated with LPS for 2 or 3 days as indicated, then stained with antibodies to CD138 and B220.

Naïve and memory B cell turnover kinetics

Next we sought to determine turnover rates for long-term memory B cell populations and establish whether these parameters differ between memory and naïve B cell subpopulations. To this end we chose to pulse immunized Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mice with DOX for approximately 3 months beginning the day after immunization. This time frame is sufficient to generate both IgM⁺ and IgG⁺ memory cells but, based on our recent data (9), might also reveal residual turnover within these populations due to persisting antigen. Therefore after 86 days of DOX treatment (pulse), we terminated exposure and evaluated frequencies of label (GFP)-retaining cells among NP-binding memory cells for an additional 45 weeks (chase; Fig. 3A).

Figure 3. Decay rates for memory and naïve B cell populations.

(A) Pulse/chase strategy for NP-CγG-immunized B6.Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mice. (B) Representative H2B-GFP expression among NP-binding IgD⁻ Dump⁻ CD19⁺ B220⁺ CD38⁺ cells at the indicated time points of the chase phase. See Figure 1A for gating strategy. (C) Mean %GFP⁺ cells among NP-binding IgD⁻ Dump⁻ CD19⁺ B220⁺ CD38⁺ cells (squares) and follicular (circles, AA4⁻ CD21⁺ CD23⁺ IgM^low) and marginal zone (triangles, AA4⁻ CD21^high CD23⁻ IgM^high) B cells. The latter two populations were gated as described (14). Data are summarized from two separate experiments with identical immunization regimens and pulse/chase schedules. For each sample at least 10 × 10⁶ events/file were collected on an LSR2 flow cytometer.

At day 0 of the chase period 85% of NP-binding CD38⁺ CD19⁺ B220⁺ memory B cells were GFP⁺ (Fig. 3B). Remarkably, although frequencies of GFP⁺ NP-binding memory cells dropped to 54–58% by day 32 of the chase period, thereafter frequencies of label retaining cells remained unchanged, even on day 317 of the chase period (Fig. 3B and C). Of note, because memory B cells may exist in one of three or more subpopulations based on differential expression of CD73, CD80, and PD-L2 (28), we did not focus on individual subsets for these analyses. However, it should also be noted that, in our hands, the vast majority of GFP⁺ and GFP⁻ NP-binding B cells detected at every time point were CD80⁺ CD73⁺ PD-L2⁺ (not shown, and see Figure 4A). The lack of measurable attrition of total numbers of memory B cells, or cellular turnover within the memory B cell pool, contrasted markedly with naïve follicular (IgM^low CD21^low CD23⁺) and marginal zone (IgM^high CD21^high CD23^low) B cells. Whereas absolute numbers of follicular and marginal zone B cells in each pool remained constant throughout these analyses (not shown), as expected frequencies of GFP⁺ cells within each population declined steadily over the entire chase period (Fig. 3C). Based on these data, we calculated half-lives for follicular and MZ B cells of 92.4 days and 153.7 days, respectively. However, due to the lack of a measurable decline in frequencies of label retaining memory B cells beyond day 32 of the chase period, we were unable to estimate the half-life of NP-specific memory B cells with any precision. Nonetheless, it is clear based on these data that the life span of memory B cells is considerably greater than their counterparts within naïve B cell populations.

Figure 4. Decay rate for IgM⁺ memory B cells.

(A) Splenocytes from an adult B6.Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mouse immunized with NP-CγG 402 days previously were stained with the indicated reagents. The left-most plot was pre-gated as shown in Figure 1A. (B) The fraction of NP-binding IgD⁻ Dump⁻ CD19⁺ B220⁺ CD38⁺ cells that were surface IgM⁺ at each time point is shown. Each symbol represents an individual mouse. (C) Representative data showing GFP expression for IgM⁺ and IgM⁻ NP-binding CD38⁺ B cells. (D) Mean %GFP⁺ cells among NP-binding IgD⁻ Dump⁻ CD19⁺ B220⁺ CD38⁺ cells that were either IgM⁺ (circles) or IgM⁻ (squares). Data are summarized from two separate experiments with identical immunization regimens and pulse/chase schedules. For each sample at least 10 × 10⁶ events/file were collected on an LSR2 flow cytometer.

Decay kinetics for IgM⁺ memory cells

Several recent studies indicate that IgM⁺ memory B cells play important roles in lasting immunity (15, 29, 30), and data from Pape et al indicate that numbers of IgM⁺ memory B cells can remain constant for upwards of 500 days post-immunization (15). Notably, Pape et al also reported that class-switched memory B cells possess a substantially shorter lifespan, as numbers of these cells declined with exponential kinetics between 100 and 400 days post-immunization in their system (15). We therefore sought to evaluate cellular turnover among IgM⁺ and IgG⁺ NP-specific memory B cell populations. Total frequencies of NP-specific IgM⁺ cells ranged from 10–40% of the total NP-specific memory B cell pool, with a slight trend towards increased representation in this population at later time points (Fig. 4A, B). However, frequencies of label-retaining IgM⁺ memory B cells declined from ~80% on day 0 to ~40% on day 73 of the chase period, but remained remarkably constant thereafter (Fig. 4C and D). In contrast, frequencies of label-retaining class-switched NP-specific B cells achieved stability much earlier, and remained constant throughout the chase period. Therefore, although the length of time needed for IgM⁺ memory B cells to achieve steady state turnover kinetics was greater compared to Ig class-switched cells, once established both IgM⁺ and IgG⁺ memory B cell pools were exceptionally stable. Therefore we conclude that both IgM⁺ and class-switched memory B cells possess exceptionally long half-lives.

Discussion

Our results show that memory and naïve B cell lifespans are radically different. Unlike naïve B cell pools, which over time experience slow but appreciable renewal from B-lineage precursors in the bone marrow, our data indicate that once generated, memory B cells persist for lengthy periods without dividing and without substantial input from newly activated cells. Due to the lack of measurable loss of label retaining cells after more than 300 days, we were unable to calculate a precise half-life for these cells. Indeed, the main constraint in estimating memory B cell lifespan was the relatively short lifespan of the mouse. If we estimate that the minimum lifespan of a memory B cell in a mouse is 30 months, or the lifespan of a C57BL/6 mouse, then the average lifespan of a memory B cell is at least 9 times greater than the average lifespan of a naïve follicular B cell, and 5.8 times that of a marginal zone B cell (900 versus 92 and 154 days, respectively). Furthermore, though many antibody-secreting plasma cells are considered to be long-lived (31), established plasma cell populations in the bone marrow may exhibit a decay rate (32), in apparent contrast to memory B cells. Therefore memory B cell populations appear to be exceptionally durable, even when compared to long-lived plasma cells.

The extensive lifespan of memory B cells raises questions about how these cells avoid apoptosis. In this regard, whereas survival of most naïve B cells requires exposure to the pro-survival cytokine BAFF/BLyS, current data suggest that BAFF is dispensable for memory B cell survival (33, 34). The differential decay rates for naïve and memory B cell pools illustrated in Figure 3 are consistent with the notion that survival of naïve and memory B cells is regulated by separate mechanisms. Further, using a similar pulse-chase regimen in Rosa26^+/rtTA, Col1A1^{+/TetOP-H2B-GFP} mice, Foudi et al. found that frequencies of GFP⁺ HSCs decline appreciably within 12 weeks due to self-renewal. By contrast, given that steady state frequencies of GFP⁺ memory B cells remained constant over >300 days, our data argue against the notion put forth originally by Fearon et al, and more recently by Luckey et al, that memory B cells self-renew in a manner similar to HSCs (10, 35). These results are also consistent with past work showing that memory cells can survive in the absence of immunizing antigen or antigen-bearing immune complexes (11, 36). Thus, it appears that neither BAFF nor persisting antigen is needed for memory B cell survival. Clearly additional work is needed for gaining an understanding of how memory B cells avoid apoptosis.

We should note that both class-switched and IgM⁺ memory pools exhibited measurable cellular turnover during the earliest phase of the chase period, with the IgM⁺ memory pool achieved steady state turnover kinetics after IgG⁺ cells (Figure 4). Given that IgM⁺ memory cells may exhibit greater dependence on BAFF than their IgG⁺ counterparts, (34), one explanation for these results is that many IgM⁺ memory B cells must compete for limiting BAFF more so than GC-derived IgG⁺ memory cells, at least during early phases of memory B cell differentiation. However, while this idea has not been tested directly, we note that IgM⁺ and IgG⁺ memory cells expressed very similar levels of TACI and BAFF-R (BR-3) before the IgM⁺ memory pool achieved steady state turnover kinetics. Alternatively, these results suggest that persisting antigen may stimulate IgM⁺ memory cell production for extended periods post-immunization. This notion is consistent with our recent observation that populations of hapten-specific IgM⁺ memory B cells exhibit residual turnover at least 52 days after immunization with NP-CγG, and that newly formed IgM-secreting plasma cells continue to colonize the bone marrow for at least 100 days post-immunization (9). It is generally thought that antigen persistence occurs through the deposition of antigen on follicular dendritic cells in GCs. Given that many IgM⁺ memory cells appear to arise without undergoing selection in GCs, these results raise questions about whether other stromal elements outside GCs can also support antigen-driven B cell differentiation. Nonetheless, our data appear to challenge the notion that, once established, memory B cell populations are routinely activated by persisting antigen or undefined ligands for toll-like receptors expressed by memory B cells to generate new plasma cells needed to maintain serum antibody concentrations (8). In this regard our data are consistent with a report by Benson and colleagues showing that non-specific stimulation of memory B cells has minimal effect of pre-existing serum antibody titers (26).

Past work suggests that memory B cells can be subdivided into several subpopulations defined by disparate degrees of somatic hypermutation and differential surface expression of CD73, PD-L2, and CD80 (20, 28). We examined expression of each of these proteins on NP-binding IgD⁻ CD38⁺ B220⁺ CD19⁺ cells at multiple time points post-immunization, and found that in every instance most of these cells were CD73⁺ PD-L2⁺ CD80⁺ (Figure 1B and not shown). However, when subdivided based on IgM versus IgG surface expression, we did detect small numbers of cells lacking at least one of these surface antigens (Figures 2 and 4A). These results contrast to some degree with those of Zuccarino-Catania et al (37), who showed that substantial numbers of NP-specific memory B cells can lack CD80 and/or PDL2. Notably, these workers further proposed that memory B cells lacking CD80 and/or PDL2 are more likely to generate GCs upon secondary immunization (37). Thus based on our data IgM⁺ memory B cells may be more likely to initiate GCs during secondary responses. Why we were unable to detect substantial numbers of memory B cells lacking CD80 and/or PDL2 is not obvious at this time. One possibility is the use of different approaches to induce and characterize NP-specific memory B cells. Whereas we utilized conventional B6 mice, most of the data supporting the existence of memory B cell subsets derive from experiments where Ig transgenic mice were employed to elevate numbers of responding B cells (20, 28). While the latter approach is necessary for experiments requiring increased cell numbers, little is known about memory B cell niches or how elevating numbers of responding B cells might alter the cellular dynamics, surface phenotype, and function of nascent and long-term memory B cells. Therefore it is conceivable that memory B cells generated in transgenic systems are more likely to encounter novel environments, and consequently acquire unique gene expression profiles and functions. Alternatively, different immunization regimens or different infectious agents may influence the degree to which distinct memory B cell subpopulations arise. Further work is needed to evaluate the influence of these and additional variables in memory B cell formation.

Whether all antigens and immunization regimens are equally effective at inducing quiescent and long-lived memory B cells remains to be tested adequately. In fact, why Pape et al (15) observed a slow but appreciable and steady decline in numbers of class switched memory B cells induced via immunization with phycoerythrin is unknown at present. Indeed, these workers also employed a different route of immunization. Some clues to this important issue might become available from studies of the generation and lifespan of antigen-experienced IgA⁺ B cells. Past data from Hapfelmeier et al. indicate that IgA⁺ B cells that form in response to colonization of germ-free mice with an auxotrophic strain of E. coli neither persist beyond a few weeks nor generate classic memory antibody responses upon re-exposure to antigen unless the gut mucosa is devoid of other commensal bacteria (38). These results suggest that the vast majority of humoral responses to commensal microbes do not result in the formation of long-lived IgA⁺ memory B cells. By contrast, earlier work involving oral immunization with cholera toxin suggests that heightened inflammatory reactions in the gut enhance the likelihood of forming lasting IgA⁺ memory cells (39–41). And quite recently Lindner et al reported a rather comprehensive analysis of the V-gene repertoire of IgA⁺ cells in the intestine suggests that many commensal microbes are also able to elicit the generation of long-lived IgA⁺ cells (42), although the latter study did not distinguish between memory B cells and plasma cells. Therefore establishing long-lived memory responses may depend heavily on numerous poorly understood variables including the antigen and TLR ligands provided by the antigen and/or the adjuvant and degree of inflammation induced.

By combining an inducible, highly stable, and innocuous in vivo labeling strategy with approaches for characterizing antigen-induced memory B cells, we have defined decay rates for pre-established class-switched and IgM⁺ memory B cell populations. Our results support the classic model wherein antigen-induced differentiation results in B cells with profoundly lengthened lifespans. Clearly, understanding how activated B cells receive and integrate signals from T cells and other sources to enact a gene expression program conducive to increased lifespan will require further investigation.