Igm Cellular Dynamics of Memory B Cell

Cellular Dynamics of Memory B Cell Populations: IgM + and IgG+ Memory B Cells Persist Indefinitely as Quiescent Cells

This information is current as Derek D. Jones, Joel R. Wilmore and David Allman of September 24, 2021. J Immunol published online 5 October 2015 http://www.jimmunol.org/content/early/2015/10/03/jimmun ol.1501365 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 5, 2015, doi:10.4049/jimmunol.1501365 The Journal of Immunology

Cellular Dynamics of Memory B Cell Populations: IgM+ and IgG+ Memory B Cells Persist Indeﬁnitely as Quiescent Cells

Derek D. Jones, Joel R. Wilmore, and David Allman

Despite their critical role in long-term immunity, the life span of individual memory B cells remains poorly deﬁned. Using a tet-

racycline-regulated pulse-chase system, we measured population turnover rates and individual t1/2 of pre-established Ag-induced Ig class-switched and IgM-positive memory B cells over 402 d. 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 t1/2 that appear to exceed the life span of the mouse, contrasting dramatically with mature naive B cells. These results indicate that recall Ab responses are mediated by stable pools of extremely long-lived cells, and suggest that Ag-experienced B cells employ remarkably efﬁcient survival mechanisms. The Journal of Immunology, 2015, 195:

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emory B cells mediate robust recall responses by vated cells, there is little information on the steady-state dynamics differentiating rapidly into Ab-secreting plasma cells of established memory B cell populations. M and forming nascent germinal centers (GCs), and by Several groups have since sought to deﬁne the life span of serving as effective APCs (1–4). Because secondary Ab responses memory B cells; however, the results have not led to a clear

can be induced long after initial Ag encounter (5), it is generally consensus. Using a BCR transgenic system, Anderson et al. (11) http://www.jimmunol.org/ assumed that individual memory B cells are exceptionally long- showed that memory B cell numbers remained constant be- lived. However, current experimental evidence for this idea is tween 8 and 20 wk postimmunization, and based on short-term varied and at times contradictory. Yet, a clear definition of memory in vivo BrdU-labeling experiments estimated the t1/2 of mem- B cell life spans and the mechanisms regulating this process is ory B cells to be 8–10 wk. Given that the accepted t1/2 of naive critical for vaccine design and for developing improved strategies B cells is 7–8 wk (12–14), based on these results it is unclear for combating Ab-mediated pathologies. whether individual memory B cells possess substantially longer In people, numbers of Ag-specific memory B cells remain life spans than their naive counterparts. By contrast, Pape et al. relatively stable for .50 y after smallpox vaccination (6). How- (15) showed immunization of conventional inbred mice with ever, these results do not provide information on the life span of the protein PE induced the generation of long-lived IgM+ and by guest on September 24, 2021 individual cells. Consequently, it is not known whether long-term class-switched (IgG+) memory cells. However, whereas in this maintenance of such populations requires periodic input from system IgM+ memory B cells remained constant for upward of activated B cells, or whether particular clones come to dominate 500 d, class-switched cells decayed with exponential kinetics, memory pools over extended time frames. The former scenario is returning to preimmunization levels by 400 d (15). Why IgM consistent with the work of Barrington et al. (7) wherein persisting and class-switched memory cells might possess distinct t1/2 Ag appeared to promote the generation of nascent memory B cells remains to be determined. These results also appear to differ well after immunization. Similarly, others have proposed that with those of Schittek and Rajewsky (16), who showed that maintenance of serum Ab titers requires the slow, but consistent class-switched memory B cell pools are relatively stable over generation of plasma cells by Ag-activated memory B cells (8, 9). 8 wk. However, the latter workers did not examine decay rates These ideas are consistent with a model put forth earlier by Fearon for extended periods, or attempt to calculate t1/2 for individual et al. (10) proposing that memory B cells employ a stem cell-like cells within this pool. self-renewal program to continuously generate plasma cells. No- To resolve these issues, we employed a nontoxic pulse-chase tably, although each of these scenarios predicts that memory B cell labeling approach. This strategy exploits a tetracycline-regulated pools contain meaningful numbers of activated or recently acti- reporter allele encoding the chromatin protein histone 2B fused to GFP. This approach allowed us to establish decay rates for individual cells within established Ag-specific memory B cell Department of Pathology and Laboratory Medicine, University of Pennsylvania populations without concern for the toxic effects associated School of Medicine, Philadelphia, PA 19104 with extended exposure to DNA nucleotide analogs such as Received for publication June 15, 2015. Accepted for publication September 14, BrdU. To provide appropriate benchmarks for this system, we 2015. also determined decay rates for naive B cell populations. Our This work was supported by National Institutes of Health Grant R01-AI097590 (to results show that whereas naive follicular and marginal zone D.A.) and National Institutes of Health Training Grant T32CA009140 (to D.D.J.). B cells exhibit decay rates consistent with a t1/2 of 13–22 wk, Address correspondence and reprint requests to Dr. David Allman, University of + + Pennsylvania, 36th Street and Hamilton Walk, 230 John Morgan Building, Philadel- decay rates for IgM and IgG memory B cells were mark- phia, PA 19104-6082. E-mail address: [email protected] edly slower, revealing cellular t1/2 greater than the 2-y life Abbreviations used in this article: BV, Brilliant Violet; DOX, doxycycline; GC, span of the mouse. These data illustrate that, once established, germinal center; NP, (4-hydroxy-3-nitrophenyl)acetyl; TACI, transmembrane activa- Ag-specific memory B cell populations are remarkably stable tor and calcium-modulating cyclophilin ligand interactor. and highly enriched for quiescent and exceptionally long-lived Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 cells.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501365 2 MEMORY B CELL LIFE SPAN

Materials and Methods ligand interactor (TACI) were PE conjugates purchased from BioLegend. Mice and immunizations Flow cytometric acquisition was performed on a BD LSRII, and analyses were performed using FlowJo 8.8.6 (Tree Star). Multiple files per sample Adult C57BL/6 or C57BL/6-backcrossed Rosa26+/rtTA,Col1A1+/TetOP-H2B-GFP were concatenated prior to data analysis. Cells were sorted for cell culture mice were purchased from Jackson Laboratories and maintained in a with a four-laser FACSAria. specific pathogen-free facility at the University of Pennsylvania, in ac- cordance with institutional guidelines for animal care and welfare. Mice Cell culture were immunized i.p. with 50 mg (4-hydroxy-3-nitrophenyl)acetyl (NP)18- Sorted B cells were cultured for 2–3 d in RPMI 1640 supplemented with chicken g-globulin in alum. To induce transgene expression, mice were 10% FCS and glutamine, antibiotics, HEPES, and 2-ME before analysis maintained on drinking water containing 2 mg/ml doxycycline (DOX; by flow cytometry. LPS (Escherichia coli) was purchased from Sigma- Sigma-Aldrich) supplemented with 10 mg/ml sucrose. Aldrich and used at 10 mg/ml.

Flow cytometry Estimation of cellular t1/2

Single-cell suspensions of splenocytes were prepared, depleted of RBCs by When possible, t1/2 for deﬁned cell populations were calculated as fol- hypotonic lysis, and stained with optimal dilutions of the indicated Abs, as lows: t1/2 =(T3 log2)/ log (starting quantity/ending quantity), where T = described (17). All of the following reagents were obtained from eBio- elapsed time. science, 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), Results anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti–Gr-1 (RB6-8C5), anti-F4/80 Inclusion of the tetracycline analog DOX in the drinking water of (BM8), and anti-TER119 (BioLegend); allophycocyanin anti-CD93 Rosa26+/rtTA, Col1A1+/TetOP-H2B-GFP mice induces the expression

(AA4.1); AlexaFluor700 anti-CD38 (90); allophycocyanin-Cy5.5 anti- of Histone2B-GFP fusion proteins. Once generated, Histone2B- Downloaded from CD19 (RM7719; Invitrogen); allophycocyanin-Cy7 anti-IgD (11-26c.2a; GFP proteins incorporate into the chromatin of most cells, BioLegend); and Brilliant Violet (BV; all from BioLegend), 605 anti-CD73 (TY/11.8), BV650 anti-CD80 (16-I0A1), BV785 anti-CD19 (6D5), and allowing the tracking of individual nondividing cells for at least biotin anti–PD-L2 (TY25; BioLegend). Biotinylated Abs were revealed 1 y after terminating DOX exposure (18). We immunized adult with streptavidin-BV421 (BioLegend). NP-allophycocyanin was conjugated B6-backcrossed Rosa26+/rtTA,Col1A1+/TetOP-H2B-GFP mice with in house, using standard methods. Dead cells were excluded from analyses immunogenic conjugates of NP conjugated to chicken g-globulin. using Zombie Aqua (BioLegend), and doublets were excluded using the

This approach allows direct identification of NP-specific B cell http://www.jimmunol.org/ combined width parameter of the forward and side scatter parameters. Abs for B cell–activating factor belonging to the TNF family (BAFF) receptor populations by flow cytometry using NP conjugates of the fluo- (BAFF-R) and transmembrane activator and calcium-modulating cyclophilin rescent protein allophycocyanin (19). Because limiting cell- by guest on September 24, 2021

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-CgG 402 d previously were stained with the indicated Abs and NP-allophycocyanin to detect NP-binding B cells. The leftmost plot was pregated for viable lymphocytes. Representative of three to five mice per time point. The rightmost 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 IgD2 Dump2 CD19+ B220+ CD38+ cells at each time point. Each circle represents a single mouse; means and SDs 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 3 106 events/file were collected on a LSR2 flow cytometer. The Journal of Immunology 3 extrinsic factors may be needed for memory B cell generation and increased surface levels of the BAFF-receptor TACI, whereas survival, we did not employ Ig transgenic mice to increase fre- levels of the primary BAFF receptor BAFF-R (or BR-3) were quencies of responding cells. To characterize Ag-experienced similar on naive and all Ag-experienced B cells examined B cells, we focused on NP-binding CD19+ IgD2 cells that also (Fig. 2B). Moreover, consistent with the notion that memory lacked the non-B cell surface Ags (“Dump”) CD4, CD8a, Gr-1, B cells generate plasma cells with increased kinetics compared F4/80, and TER-119 (19). We also focused on B220+ CD38+ cells with naive B cells (26, 27), when stimulated with LPS NP- to avoid persisting GC B cells (2). With this strategy, we were able binding IgD2 CD38+ CD19+ cells generated CD138+ plasma to detect NP-specific B cells for at least 402 d postimmunization cells faster and more effectively than naive follicular B cells (Fig. 1A, 1C). The vast majority of NP-binding CD38+ CD19+ (Fig. 2C). Altogether, these results show that NP-specific memory B220+ (Dump2) B cells uniformly expressed the memory B cell B cells achieve steady state within 86 d postimmunization and can surface phenotype CD73+ PD-L2+ at every time point (Fig. 1A, be detected for extended periods postimmunization without em- 1B; data not shown) (20). Consistent with several reports show- ploying Ig transgenic mice. ing that Igl+ B cells dominate the NP-specific Ab response in B6 mice (21–23), the majority of NP-binding cells were Igl+;yetit Naive and memory B cell turnover kinetics should be emphasized that some 30% of these cells were Igk+ Next we sought to determine turnover rates for long-term memory (Fig. 1B). B cell populations and establish whether these parameters differ Notably, absolute numbers of NP-binding CD38+ CD19+ between memory and naive B cell subpopulations. To this end, we B220+ cells remained constant between 86 and 402 d post- chose to pulse immunized Rosa26+/rtTA, Col1A1+/TetOP-H2B-GFP immunization (Fig. 1C). Furthermore, Ag-induced NP-binding mice with DOX for ∼3 mo beginning the day after immunization. Downloaded from B cells defined by these criteria could be readily subdivided This time frame is sufficient to generate both IgM+ and IgG+ into either IgM+ or IgG+ subpopulations, and both fractions were memory cells but, based on our recent data (9), might also reveal highly enriched for CD73+ PD-L2+ cells (Fig. 2A). Therefore, residual turnover within these populations due to persisting Ag. this strategy allowed us to identify both IgM+ and class-switched Therefore, after 86 d of DOX treatment (pulse), we terminated memory B cells. Consistent with this conclusion, as shown pre- exposure and evaluated frequencies of label (GFP)-retaining cells

viously for bulk memory B cells in mice and humans (24, 25), among NP-binding memory cells for an additional 45 wk (chase; http://www.jimmunol.org/ both IgM+ and IgG+ Ag-induced NP-binding cells expressed Fig. 3A). by guest on September 24, 2021

FIGURE 2. Characteristics of IgM+ and IgG+ NP-specific memory B cells. (A) Splenocytes from a C57BL/6 adult immunized with NP-CgG54d previously were stained with the indicated reagents. The leftmost plot was pregated as shown in Fig. 1A. A total of 10 3 106 events was collected on a LSR2 flow cytometer. (B) Splenocytes from a C57BL/6 mouse immunized with NP-CgG 117 d previously were stained, as shown in the leftmost plot in (A), and with either anti–BAFF-R or anti-TACI Abs. A total of 10 3 106 events was analyzed. Background staining (filled gray histogram) was established by gating on Dump+ cells (data not shown). (C) NP-binding IgD2 Dump2 CD19+ B220+ CD38+ cells harvested from C57BL/6 mice 117 d postimmunization, or naive follicular (CD19+ CD23+ CD21int AA42) or marginal zone (CD19+ CD232 CD21high AA42) B cells sorted from naive adults were stimulated with LPS for 2 or 3 d as indicated, and then stained with Abs to CD138 and B220. 4 MEMORY B CELL LIFE SPAN Downloaded from http://www.jimmunol.org/

FIGURE 3. Decay rates for memory and naive B cell populations. (A) Pulse/chase strategy for NP-CgG–immunized B6.Rosa26+/rtTA,Col1A1+/TetOP-H2B-GFP mice. (B) Representative H2B-GFP expression among NP-binding IgD2 Dump2 CD19+ B220+ CD38+ cells at the indicated time points of the chase phase. See Fig. 1A for gating strategy. (C)Mean%GFP+ cells among NP-binding IgD2 Dump2 CD19+ B220+ CD38+ cells (squares) and follicular (circles, AA42 CD21+ CD23+ IgMlow) and marginal zone (triangles, AA42 CD21high CD232 IgMhigh) 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 3 106 events/ﬁle were collected on a LSR2 ﬂow cytometer.

At day 0 of the chase period, 85% of NP-binding CD38+ CD19+ Pape et al. (15) indicate that numbers of IgM+ memory B cells can by guest on September 24, 2021 B220+ memory B cells were GFP+ (Fig. 3B). Remarkably, although remain constant for upward of 500 d postimmunization. Notably, frequencies of GFP+ NP-binding memory cells dropped to 54–58% Pape et al. (15) also reported that class-switched memory B cells by day 32 of the chase period, thereafter frequencies of label- possess a substantially shorter life span, as numbers of these cells retaining cells remained unchanged, even on day 317 of the chase declined with exponential kinetics between 100 and 400 d post- period (Fig. 3B, 3C). Of note, because memory B cells may exist in immunization in their system. We therefore sought to evaluate one of three or more subpopulations based on differential expres- cellular turnover among IgM+ and IgG+ NP-specific memory sion of CD73, CD80, and PD-L2 (28), we did not focus on indi- B cell populations. Total frequencies of NP-specific IgM+ cells vidual subsets for these analyses. However, it should also be noted ranged from 10 to 40% of the total NP-specific memory B cell that, in our hands, the vast majority of GFP+ and GFP2 NP-binding pool, with a slight trend toward increased representation in this B cells detected at every time point were CD80+ CD73+ PD-L2+ population at later time points (Fig. 4A, 4B). However, frequen- (data not shown; see Fig. 4A). The lack of measurable attrition of cies of label-retaining IgM+ memory B cells declined from ∼80% total numbers of memory B cells, or cellular turnover within the on day 0 to ∼40% on day 73 of the chase period, but remained memory B cell pool, contrasted markedly with naive follicular remarkably constant thereafter (Fig. 4C, 4D). In contrast, fre- (IgMlow CD21low CD23+) and marginal zone (IgMhigh CD21high quencies of label-retaining class-switched NP-specific B cells CD23low) B cells. Whereas absolute numbers of follicular and achieved stability much earlier and remained constant through- marginal zone B cells in each pool remained constant throughout out the chase period. Therefore, although the length of time these analyses (data not shown), as expected, frequencies of GFP+ needed for IgM+ memory B cells to achieve steady-state turnover cells within each population declined steadily over the entire chase kinetics was greater compared with Ig class-switched cells, once + + period (Fig. 3C). Based on these data, we calculated t1/2 for fol- established, both IgM and IgG memory B cell pools were ex- licular and MZ B cells of 92.4 and 153.7 d, respectively. However, ceptionally stable. Therefore, we conclude that both IgM+ and due to the lack of a measurable decline in frequencies of label- class-switched memory B cells possess exceptionally long t1/2. retaining memory B cells beyond day 32 of the chase period, we were unable to estimate the t1/2 of NP-specific memory B cells with Discussion any precision. Nonetheless, it is clear based on these data that the Our results show that memory and naive B cell life spans are life span of memory B cells is considerably greater than their radically different. Unlike naive B cell pools, which over time ex- counterparts within naive B cell populations. perience slow but appreciable renewal from B-lineage precursors in

+ the bone marrow, our data indicate that, once generated, memory Decay kinetics for IgM memory cells B cells persist for lengthy periods without dividing and without Several recent studies indicate that IgM+ memory B cells play substantial input from newly activated cells. Due to the lack of important roles in lasting immunity (15, 29, 30), and data from measurable loss of label-retaining cells after .300 d, we were The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

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- CgG 402 d previously were stained with the indicated reagents. The leftmost plot was pregated, as shown in Fig. 1A. (B) The fraction of NP-binding IgD2 by guest on September 24, 2021 Dump2 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 IgM2 NP-binding CD38+ B cells. (D) Mean %GFP+ cells among NP-binding IgD2 Dump2 CD19+ B220+ CD38+ cells that were either IgM+ (circles) or IgM2 (squares). Data are summarized from two separate experiments with identical immunization regimens and pulse/chase schedules. For each sample, at least 10 3 106 events/ﬁle were collected on a LSR2 ﬂow cytometer.

unable to calculate a precise t1/2 for these cells. Indeed, the main B cells remained constant over .300 d, our data argue against the constraint in estimating memory B cell life span was the relatively notion put forth originally by Fearon et al. (10) and more recently short life span of the mouse. If we estimate that the minimum life by Luckey et al. (35) that memory B cells self-renew in a manner span of a memory B cell in a mouse is 30 mo, or the life span of similar to hemopoietic stem cells. These results are also consistent a C57BL/6 mouse, then the average life span of a memory B cell with past work showing that memory cells can survive in the is at least 9 times greater than the average life span of a naive absence of immunizing Ag or Ag-bearing immune complexes (11, follicular B cell, and 5.8 times that of a marginal zone B cell (900 36). Thus, it appears that neither BAFF nor persisting Ag is versus 92 and 154 d, respectively). Furthermore, although many needed for memory B cell survival. Clearly, additional work is Ab-secreting plasma cells are considered to be long-lived (31), needed for gaining an understanding of how memory B cells avoid established plasma cell populations in the bone marrow may ex- apoptosis. hibit a decay rate (32), in apparent contrast to memory B cells. We should note that both class-switched and IgM+ memory Therefore, memory B cell populations appear to be exceptionally pools exhibited measurable cellular turnover during the earliest durable, even when compared with long-lived plasma cells. phase of the chase period, with the IgM+ memory pool achieving The extensive life span of memory B cells raises questions about steady-state turnover kinetics after IgG+ cells (Fig. 4). Given that how these cells avoid apoptosis. In this regard, whereas survival of IgM+ memory cells may exhibit greater dependence on BAFF than most naive B cells requires exposure to the prosurvival cytokine their IgG+ counterparts (34), one explanation for these results is BAFF/BLyS, current data suggest that BAFF is dispensable for that many IgM+ memory B cells must compete for limiting BAFF memory B cell survival (33, 34). The differential decay rates for more so than GC-derived IgG+ memory cells, at least during early naive and memory B cell pools illustrated in Fig. 3 are consistent phases of memory B cell differentiation. However, although this with the notion that survival of naive and memory B cells is idea has not been tested directly, we note that IgM+ and IgG+ regulated by separate mechanisms. Furthermore, using a similar memory cells expressed very similar levels of TACI and BAFF-R pulse-chase regimen in Rosa26+/rtTA, Col1A1+/TetOP-H2B-GFP mice, (BR-3) before the IgM+ memory pool achieved steady-state Foudi et al. (18) found that frequencies of GFP+ hemopoietic stem turnover kinetics. Alternatively, these results suggest that per- cells decline appreciably within 12 wk due to self-renewal. By sisting Ag may stimulate IgM+ memory cell production for ex- contrast, given that steady-state frequencies of GFP+ memory tended periods postimmunization. This notion is consistent with 6 MEMORY B CELL LIFE SPAN our recent observation that populations of hapten-specific IgM+ data from Hapfelmeier et al. (38) indicate that IgA+ B cells that memory B cells exhibit residual turnover at least 52 d after im- form in response to colonization of germ-free mice with an munization with NP-CgG, and that newly formed IgM-secreting auxotrophic strain of E. coli neither persist beyond a few weeks plasma cells continue to colonize the bone marrow for at least nor generate classic memory Ab responses upon re-exposure to 100 d postimmunization (9). It is generally thought that Ag per- Ag unless the gut mucosa is devoid of other commensal bacteria. sistence occurs through the deposition of Ag on follicular den- These results suggest that the vast majority of humoral responses dritic cells in GCs. Given that many IgM+ memory cells appear to to commensal microbes do not result in the formation of long- arise without undergoing selection in GCs, these results raise lived IgA+ memory B cells. By contrast, earlier work involving questions about whether other stromal elements outside GCs can oral immunization with cholera toxin suggests that heightened also support Ag-driven B cell differentiation. Nonetheless, our inflammatory reactions in the gut enhance the likelihood of data appear to challenge the notion that, once established, mem- forming lasting IgA+ memory cells (39–41). And quite recently, ory B cell populations are routinely activated by persisting Ag Lindner et al. (42) reported a rather comprehensive analysis of or undefined ligands for TLRs expressed by memory B cells the V-gene repertoire of IgA+ cells in the intestine suggests that to generate new plasma cells needed to maintain serum Ab con- many commensal microbes are also able to elicit the generation centrations (8). In this regard, our data are consistent with of long-lived IgA+ cells, although the latter study did not dis- a report by Benson et al. (26) showing that nonspecific stim- tinguish between memory B cells and plasma cells. Therefore, ulation of memory B cells has minimal effect of pre-existing establishing long-lived memory responses may depend heavily serum Ab titers. on numerous poorly understood variables, including the Ag and

Past work suggests that memory B cells can be subdivided into TLR ligands provided by the Ag and/or the adjuvant and degree Downloaded from several subpopulations defined by disparate degrees of somatic of inflammation induced. hypermutation and differential surface expression of CD73, By combining an inducible, highly stable, and innocuous in vivo PD-L2, and CD80 (20, 28). We examined expression of each of labeling strategy with approaches for characterizing Ag-induced these proteins on NP-binding IgD2 CD38+ B220+ CD19+ cells at memory B cells, we have defined decay rates for pre-established multiple time points postimmunization, and found that in every class-switched and IgM+ memory B cell populations. Our results + + + instance most of these cells were CD73 PD-L2 CD80 (Fig. 1B; support the classic model wherein Ag-induced differentiation http://www.jimmunol.org/ data not shown). However, when subdivided based on IgM versus results in B cells with profoundly lengthened life spans. Clearly, IgG surface expression, we did detect small numbers of cells understanding how activated B cells receive and integrate signals lacking at least one of these surface Ags (Figs. 2, 4A). These from T cells and other sources to enact a gene expression program results contrast to some degree with those of Zuccarino-Catania conducive to increased life span will require further investigation. et al. (37), who showed that substantial numbers of NP-specific memory B cells can lack CD80 and/or PD-L2. Notably, these Acknowledgments workers further proposed that memory B cells lacking CD80 We thank Drs. Michael Cancro and Uri Hershberg for helpful discussions. and/or PD-L2 are more likely to generate GCs upon secondary immunization (37). Thus, based on our data, IgM+ memory B cells by guest on September 24, 2021 may be more likely to initiate GCs during secondary responses. Disclosures Why we were unable to detect substantial numbers of memory The authors have no financial conflicts of interest. B cells lacking CD80 and/or PD-L2 is not obvious at this time. One possibility is the use of different approaches to induce and References characterize NP-specific memory B cells. Whereas we used con- 1. Good-Jacobson, K. L., and M. J. Shlomchik. 2010. Plasticity and heterogeneity ventional B6 mice, most of the data supporting the existence of in the generation of memory B cells and long-lived plasma cells: the influence of memory B cell subsets derive from experiments in which Ig germinal center interactions and dynamics. J. Immunol. 185: 3117–3125. 2. Dogan, I., B. Bertocci, V. Vilmont, F. Delbos, J. Me´gret, S. Storck, transgenic mice were employed to elevate numbers of responding C. A. Reynaud, and J. C. Weill. 2009. Multiple layers of B cell memory with B cells (20, 28). Although the latter approach is necessary for different effector functions. Nat. Immunol. 10: 1292–1299. experiments requiring increased cell numbers, little is known 3. Good-Jacobson, K. L., E. Song, S. Anderson, A. H. Sharpe, and M. J. Shlomchik. 2012. CD80 expression on B cells regulates murine T follicular about memory B cell niches or how elevating numbers of helper development, germinal center B cell survival, and plasma cell generation. responding B cells might alter the cellular dynamics, surface J. Immunol. 188: 4217–4225. 4. McHeyzer-Williams, L. J., P. J. Milpied, S. L. Okitsu, and M. G. McHeyzer- phenotype, and function of nascent and long-term memory Williams. 2015. Class-switched memory B cells remodel BCRs within second- B cells. Therefore, it is conceivable that memory B cells generated ary germinal centers. Nat. Immunol. 16: 296–305. in transgenic systems are more likely to encounter novel envi- 5. Gowans, J. L., and J. W. Uhr. 1966. The carriage of immunological memory by small lymphocytes in the rat. J. Exp. Med. 124: 1017–1030. ronments, and consequently acquire unique gene expression pro- 6. Crotty, S., P. Felgner, H. Davies, J. Glidewell, L. Villarreal, and R. Ahmed. 2003. files and functions. Alternatively, different immunization regimens Cutting edge: long-term B cell memory in humans after smallpox vaccination. J. or different infectious agents may influence the degree to which Immunol. 171: 4969–4973. 7. Barrington, R. A., O. Pozdnyakova, M. R. Zafari, C. D. Benjamin, and distinct memory B cell subpopulations arise. Further work is M. C. Carroll. 2002. B lymphocyte memory: role of stromal cell complement needed to evaluate the influence of these and additional variables and FcgammaRIIB receptors. J. Exp. Med. 196: 1189–1199. 8. Bernasconi, N. L., E. Traggiai, and A. Lanzavecchia. 2002. Maintenance of in memory B cell formation. serological memory by polyclonal activation of human memory B cells. Science Whether all Ags and immunization regimens are equally ef- 298: 2199–2202. fective at inducing quiescent and long-lived memory B cells 9. Chernova, I., D. D. Jones, J. R. Wilmore, A. Bortnick, M. Yucel, U. Hershberg, and D. Allman. 2014. Lasting antibody responses are mediated by a combination remains to be tested adequately. In fact, why Pape et al. (15) of newly formed and established bone marrow plasma cells drawn from clonally observed a slow but appreciable and steady decline in numbers distinct precursors. J. Immunol. 193: 4971–4979. of class-switched memory B cells induced via immunization 10. Fearon, D. T., P. Manders, and S. D. Wagner. 2001. Arrested differentiation, the self-renewing memory lymphocyte, and vaccination. Science 293: 248–250. with PE is unknown at present. Indeed, these workers also 11. Anderson, S. M., L. G. Hannum, and M. J. Shlomchik. 2006. Memory B cell employed a different route of immunization. Some clues to this survival and function in the absence of secreted antibody and immune complexes on follicular dendritic cells. J. Immunol. 176: 4515–4519. important issue might become available from studies of the 12. Sprent, J., and A. Basten. 1973. Circulating T and B lymphocytes of the mouse. + generation and life span of Ag-experienced IgA B cells. Past II. Lifespan. Cell. Immunol. 7: 40–59. The Journal of Immunology 7

13. Allman, D. M., S. E. Ferguson, V. M. Lentz, and M. P. Cancro. 1993. Peripheral 29. Taylor, J. J., K. A. Pape, and M. K. Jenkins. 2012. A germinal center- B cell maturation. II. Heat-stable antigen(hi) splenic B cells are an immature independent pathway generates unswitched memory B cells early in the pri- developmental intermediate in the production of long-lived marrow-derived mary response. J. Exp. Med. 209: 597–606. B cells. J. Immunol. 151: 4431–4444. 30. Kaji, T., A. Ishige, M. Hikida, J. Taka, A. Hijikata, M. Kubo, T. Nagashima, 14. Srivastava, B., W. J. Quinn, III, K. Hazard, J. Erikson, and D. Allman. 2005. Y. Takahashi, T. Kurosaki, M. Okada, et al. 2012. Distinct cellular pathways Characterization of marginal zone B cell precursors. J. Exp. Med. 202: 1225–1234. select germline-encoded and somatically mutated antibodies into immunological 15. Pape, K. A., J. J. Taylor, R. W. Maul, P. J. Gearhart, and M. K. Jenkins. 2011. memory. J. Exp. Med. 209: 2079–2097. Different B cell populations mediate early and late memory during an endoge- 31. Manz, R. A., A. Thiel, and A. Radbruch. 1997. Lifetime of plasma cells in the nous immune response. Science 331: 1203–1207. bone marrow. Nature 388: 133–134. 16. Schittek, B., and K. Rajewsky. 1990. Maintenance of B-cell memory by long- 32. Slifka, M. K., R. Antia, J. K. Whitmire, and R. Ahmed. 1998. Humoral immunity lived cells generated from proliferating precursors. Nature 346: 749–751. due to long-lived plasma cells. Immunity 8: 363–372. 17. Calamito, M., M. M. Juntilla, M. Thomas, D. L. Northrup, J. Rathmell, 33.Benson,M.J.,S.R.Dillon,E.Castigli,R.S.Geha,S.Xu,K.P.Lam,and M. J. Birnbaum, G. Koretzky, and D. Allman. 2010. Akt1 and Akt2 promote R. J. Noelle. 2008. Cutting edge: the dependence of plasma cells and inde- peripheral B-cell maturation and survival. Blood 115: 4043–4050. pendence of memory B cells on BAFF and APRIL. J. Immunol. 180: 3655– 18. Foudi, A., K. Hochedlinger, D. Van Buren, J. W. Schindler, R. Jaenisch, 3659. V. Carey, and H. Hock. 2009. Analysis of histone 2B-GFP retention reveals 34. Scholz, J. L., J. E. Crowley, M. M. Tomayko, N. Steinel, P. J. O’Neill, slowly cycling hematopoietic stem cells. Nat. Biotechnol. 27: 84–90. W. J. Quinn, III, R. Goenka, J. P. Miller, Y. H. Cho, V. Long, et al. 2008. BLyS 19. McHeyzer-Williams, M. G., M. J. McLean, P. A. Lalor, and G. J. Nossal. 1993. inhibition eliminates primary B cells but leaves natural and acquired humoral Antigen-driven B cell differentiation in vivo. J. Exp. Med. 178: 295–307. immunity intact. Proc. Natl. Acad. Sci. USA 105: 15517–15522. 20. Anderson, S. M., M. M. Tomayko, A. Ahuja, A. M. Haberman, and 35. Luckey, C. J., D. Bhattacharya, A. W. Goldrath, I. L. Weissman, C. Benoist, and M. J. Shlomchik. 2007. New markers for murine memory B cells that deﬁne D. Mathis. 2006. Memory T and memory B cells share a transcriptional program mutated and unmutated subsets. J. Exp. Med. 204: 2103–2114. of self-renewal with long-term hematopoietic stem cells. Proc. Natl. Acad. Sci. 21. Jack, R. S., T. Imanishi-Kari, and K. Rajewsky. 1977. Idiotypic analysis of the USA 103: 3304–3309. response of C57BL/6 mice to the (4-hydroxy-3-nitrophenyl)acetyl group. Eur. J. 36. Maruyama, M., K. P. Lam, and K. Rajewsky. 2000. Memory B-cell persistence is Immunol. 7: 559–565. independent of persisting immunizing antigen. Nature 407: 636–642. 22. Jacob, J., R. Kassir, and G. Kelsoe. 1991. In situ studies of the primary immune 37. Zuccarino-Catania, G. V., S. Sadanand, F. J. Weisel, M. M. Tomayko, H. Meng, Downloaded from response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of S. H. Kleinstein, K. L. Good-Jacobson, and M. J. Shlomchik. 2014. CD80 and responding cell populations. J. Exp. Med. 173: 1165–1175. PD-L2 deﬁne functionally distinct memory B cell subsets that are independent of 23. Reth, M., G. J. Ha¨mmerling, and K. Rajewsky. 1978. Analysis of the repertoire of antibody isotype. Nat. Immunol. 15: 631–637. anti-NP antibodies in C57BL/6 mice by cell fusion. I. Characterization of antibody 38. Hapfelmeier, S., M. A. Lawson, E. Slack, J. K. Kirundi, M. Stoel, families in the primary and hyperimmune response. Eur. J. Immunol. 8: 393–400. M. Heikenwalder, J. Cahenzli, Y. Velykoredko, M. L. Balmer, K. Endt, et al. 24. Yates, J. L., R. Racine, K. M. McBride, and G. M. Winslow. 2013. T cell- 2010. Reversible microbial colonization of germ-free mice reveals the dynamics dependent IgM memory B cells generated during bacterial infection are re- of IgA immune responses. Science 328: 1705–1709.

quired for IgG responses to antigen challenge. J. Immunol. 191: 1240–1249. 39. Lycke, N., and J. Holmgren. 1989. Adoptive transfer of gut mucosal antitoxin http://www.jimmunol.org/ 25. Darce, J. R., B. K. Arendt, S. K. Chang, and D. F. Jelinek. 2007. Divergent memory by isolated B cells 1 year after oral immunization with cholera toxin. effects of BAFF on human memory B cell differentiation into Ig-secreting cells. Infect. Immun. 57: 1137–1141. J. Immunol. 178: 5612–5622. 40. Vajdy, M., and N. Lycke. 1993. Stimulation of antigen-specific T- and B-cell 26. Benson, M. J., R. Elgueta, W. Schpero, M. Molloy, W. Zhang, E. Usherwood, memory in local as well as systemic lymphoid tissues following oral immuni- and R. J. Noelle. 2009. Distinction of the memory B cell response to cognate zation with cholera toxin adjuvant. Immunology 80: 197–203. antigen versus bystander inflammatory signals. J. Exp. Med. 206: 2013–2025. 41. Lycke, N., and M. Bemark. 2010. Mucosal adjuvants and long-term memory 27. Zhang, J., Y. J. Liu, I. C. MacLennan, D. Gray, and P. J. Lane. 1988. B cell development with special focus on CTA1-DD and other ADP-ribosylating tox- memory to thymus-independent antigens type 1 and type 2: the role of lipo- ins. Mucosal Immunol. 3: 556–566. polysaccharide in B memory induction. Eur. J. Immunol. 18: 1417–1424. 42. Lindner, C., I. Thomsen, B. Wahl, M. Ugur, M. K. Sethi, M. Friedrichsen, 28. Tomayko, M. M., N. C. Steinel, S. M. Anderson, and M. J. Shlomchik. 2010. A. Smoczek, S. Ott, U. Baumann, S. Suerbaum, et al. 2015. Diversification of Cutting edge: hierarchy of maturity of murine memory B cell subsets. J. memory B cells drives the continuous adaptation of secretory antibodies to gut

Immunol. 185: 7146–7150. microbiota. Nat. Immunol. 16: 880–888. by guest on September 24, 2021