Germinal Center Interactions and Dynamics This Information Is Current As of September 23, 2021

Plasticity and Heterogeneity in the Generation of Memory B Cells and Long-Lived Plasma Cells: The Influence of Germinal Center Interactions and Dynamics This information is current as of September 23, 2021. Kim L. Good-Jacobson and Mark J. Shlomchik J Immunol 2010; 185:3117-3125; ; doi: 10.4049/jimmunol.1001155 http://www.jimmunol.org/content/185/6/3117 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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Plasticity and Heterogeneity in the Generation of Memory B Cells and Long-Lived Plasma Cells: The Influence of Germinal Center Interactions and Dynamics Kim L. Good-Jacobson*,† and Mark J. Shlomchik*,† In the humoral response, short-lived plasmablasts gener- formation or fully understand what regulates the quality and ate an early burst of Ab that probably plays an initial pro- quantity of the memory and PC populations. Why do different tective role. Simultaneously, another arm of the response infections or types of vaccines generate different lengths and is often triggered that leads to delayed effector function qualities of immune protection? How is the Bmem cell pop- but long-term protection. This arm comprises the germi- ulation maintained in the face of recurring infections? Are there different types of memory cells geared to be functionally dif- nal center response and its products: long-lived memory Downloaded from ferent? Addressing these questions and gaining a deeper un- B(Bmem) cells and plasma cells (PCs). The factors that derstanding of B cell memory will likely lead to a better control the differentiation of PCs and Bmem cells, as well as the composition and function of the memory com- understanding of the success of vaccines to different pathogens partment—how it self-renews while generating rapid and, therefore, will inform vaccine design in the future. secondary effector function—are poorly understood. The most basic definition of a Bmem cell population is that it is Ag experienced and persists in the absence of the immu- Recent work in mice and humans is beginning to illu- http://www.jimmunol.org/ nizing agent. Two other qualities often attributed to Bmem cells minate these issues. We review this progress, with em- are that, compared with naive cells, they respond more rapidly phasis on events in the germinal center, especially B–T during a secondary response and they have a higher affinity for interactions, which influence the development of mem- Ag. However, the latter attribute is associated with cells that ory and PC compartments and on Bmem cell heterogene- have participated in a germinal center (GC) reaction, sug- ity that may underlie flexibility and self-renewal of long- gesting that GC B cells are precursors to Bmem cells. Although lived humoral immunity. The Journal of Immunology, it seems clear that GC cells can be precursors to Bmem cells, it is 2010, 185: 3117–3125. uncertain whether all Bmem cells have derived from GC pre- cursors. by guest on September 23, 2021 he major function of the primary immune response is to generate a robust and appropriate effector response to GC-derived long-lived populations of Ag-experienced cells T any foreign stimulus. In principle, the nature of this There are two broad types of humoral responses: T independent response has evolved to successfully eliminate foreign pathogens (TI) and T dependent (TD). Although TI responses can gen- while sparing the host excessive collateral damage. To ac- erate abortive GCs (2, 3), TD responses typically are associated complish this, the immune system must balance producing with fully developed GCs. In the TD GC, Ag-specific B cells a large number of effector cells targeting foreign Ags while undergo numerous rounds of division and can undergo somatic safeguarding host cells by appropriately regulating and ulti- hypermutation (SHM), class-switch recombination, and affin- mately turning off the response. In addition to effector cells, ity selection (4–7). Therefore, the GC response endows this the primary response produces Ag-specific memory cells that population of Ag-activated B cells with qualities, such as higher remain after the infection has been cleared. These cells can affinity for the Ag and a relevant Ig isotype, which should result be reactivated upon re-exposure to the same Ag, rapidly differ- in more efficient clearance of Ag. However, the GC itself is entiating into effector cells. In the humoral response, memory transient, and GC cells themselves are not known to mediate B(Bmem) cells, together with long-lived plasma cells (PCs), effector functions. Instead, some GC B cells differentiate into enhance host protection for long periods of time. PCs or Bmem cells, both of which persist after the primary re- Although we understand the basic features of a humoral re- sponse has subsided and directly or indirectly provide effector sponse (1), we still do not know the signals that invoke Bmem cell function.

† *Department of Laboratory Medicine and Department of Immunobiology, Yale Uni- Abbreviations used in this paper: AFC, Ab-forming cell; Bcl, B cell lymphoma; BLyS, versity, New Haven, CT 06519 B lymphocyte stimulator; Bmem, memory B; FDC, follicular dendritic cell; GC, germinal center; NP, (4-hydroxy-3-nitrophenyl)acetyl; PC, plasma cell; PD-1, programmed death-1; Received for publication June 17, 2010. Accepted for publication July 27, 2010. PNA, peanut agglutinin; SAP, signaling lymphocytic activation molecule-associated pro- This work was supported by National Institutes of Health Grant AI43603 (to M.J.S.). tein; SHM, somatic hypermutation; TD, T dependent; TFH, T follicular helper cell; Tg, K.L.G-J. was supported by a National Health and Medical Research Council Biomedical transgenic; TI, T independent; WT, wild-type. Research Fellowship and an Arthritis Australia Postdoctoral Fellowship. Ó Address correspondence and reprint requests to Dr. Mark J. Shlomchik, Department of Copyright 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Laboratory Medicine, Yale University School of Medicine, P.O. Box 208035, New Haven, CT 06520-8035. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001155 3118 BRIEF REVIEWS: INFLUENCE OF GC DYNAMICS ON B CELL MEMORY

In contrast to the more open-ended definition given above, One hypothesis is that a master regulator of transcription directs the classical description of Bmem cells requires that they have the fate of a Bmem cell. B cell lymphoma (Bcl)-6 is required to undergone affinity maturation and selection. This led research- induce the GC transcriptional program (54), and B lymphocyte- ers to conclude that the GC environment is necessary for induced maturation protein-1 plays a similar role in PCs (55). Bmem cell formation. Unfortunately, the arguments supporting However, despite extensive investigation of gene-expression pro- GCs as the sole location in which Bmem cells are formed can be files of human and murine Bmem cells (56–59), no single de- circular (reviewed in Refs. 8 and 9). Nevertheless, numerous terministic transcription factor for Bmem cells has come to light. studies demonstrated that in certain immunodeficiencies in In fact, unlike the comparison between naive cells and GC which a functional GC response is abrogated, the Bmem cell B cells or PCs, the transcriptomes of naive and Bmem cells are pool is altered in number, affinity maturation, and/or ability to very similar (56–59). generate secondary responses. These immunodeficiencies are There have been a number of proposals for factors that may also marked by a decrease in circulating Ab and long-lived PC determine Bmem cell differentiation. Researchers suggested that numbers (Table I). Therefore, although it is likely that long- Bcl-6 (56) or CD40 (57) can regulate Bmem cell fate decisions, lived cells can be formed in the absence of functional GCs, the although these proposals have not been borne out (58). Al- GC microenvironment plays an important role in producing ternatively, affinity selection, in some cases by follicular den- a classically defined, fully formed Bmem cell population, as well dritic cell (FDC)-bound immune complexes, has also been as long-lived PCs. suggested to determine the fate of B cells, with high-

affinity cells directed to differentiate into PCs (63, 64). How- Downloaded from The role of T cells in GC responses ever, subsequent studies demonstrated that higher-affinity Under normal circumstances, fully developed GCs require T cell cells were undergoing more expansion early in the response, function. B cell–T cell interactions occur throughout the hu- rather than being actively directed into the PC-differentiation moral response, either at the border between the T and B cell pathway (65). Furthermore, in the absence of immune com- zones or within the follicle itself, and the nature of these inter- plexes, GC responses and Bmem cells still form robustly (66); actions changes over time. Several such interactions and sub- therefore, signals from immune complexes on FDCs are likely http://www.jimmunol.org/ sequent signaling are vital in the early stages of the response. In not involved in the fate decision per se. the absence of signaling downstream of CD40 or ICOS, for Given the failure to find deterministic signals that direct example, GC initiation is disrupted. Conversely, in the absence memory cell formation from a GC precursor, it is possible that of B cells, T cells in the follicle (T follicular helper cells [TFHs]) such a singular directive does not exist. Hodgkin and colleagues are also absent (38, 46). It remains unclear whether the absence (67–69) hypothesized, based on a series of in vitro experiments of TFHs in B cell-deficient animals is due to a complete inhibi- and computer models, that a uniform population of B cells is tion of differentiation into TFHs or whether differentiation can intrinsically able to commit to any one of several different fates occur, but the phenotype of the TFH subset requires B cells for postactivation (such as division, differentiation, or death) based by guest on September 23, 2021 maintenance (47). In this vein, the absence of signaling lympho- on stochastic factors, influenced by integrated external and cytic activation molecule-associated protein (SAP), which is ex- intrinsic signals. For example, B cells stimulated with CD40 pressed on T cells, directly impacted the duration of B cell–T cell will not all behave in the same manner. Some remain un- interactions (48), leading to diminished TFH development and divided, some divide, and a proportion differentiate. Hodgkin expansion. Therefore, when the quality of signaling downstream and colleagues proposed that at each division, some cells of early B–T interactions is impaired, formation of GCs is also commit to each of these fates, but changing intrinsic or ex- disrupted. trinsic factors in the response only affect the probability of one After a GC has formed, TFHs critically affect the outcome. event occurring over the other. In this model, the ability of TFHs influence B cells via two mechanisms: direct cell contact a B cell to differentiate is part of its cellular make-up and can be and cytokine secretion. TFHs can control the initiation and ter- influenced in a probabilistic sense, but not determined, by mination of humoral responses through the expression of posi- extrinsic stimuli. tive and inhibitory ligands and receptors, such as CD40L, ICOS, In the case of the GC, we suggest that T cell-derived signals programmed death-1 (PD-1), CD28, and CTLA-4, thereby reg- regulate the quantity and quality of the Bmem cell population by ulating the extent of the response and possibly the quantity of influencing B cell behavior. The role of signals in producing effector cells (10, 11, 13–15, 17, 23, 49). TFHs also affect the a fully formed Bmem cell population can be subdivided into quality and types of B cells produced (e.g., via the well-document those that are important for GC homeostasis and those that effects of T cell-derived cytokines in influencing B cell survival, may influence B cell differentiation itself. Indeed, a number of proliferation, isotype switching, and differentiation) (27, 50–53). recent publications investigated the effects of specific genetic In addition, the availability of T cell help and the expression of and/or cellular defects on GC B cell dynamics (progression, FasL (CD178) regulate selection within the GC, affecting the cell death, and size), as well as differentiative outputs of GCs affinity of resulting long-lived PCs (28, 33, 52). Although it is (i.e., Bmem cells and PCs). evident from these studies that T cells can affect GC dynamics, are they involved in the decision of GC B cells to differentiate Dynamics of GCs and the effect on long-lived populations into Bmem cells or PCs? In this regard, it is important to recognize that the output from the GC seems to change over time. At early stages, the How is Bmem cell and Ab-forming cell differentiation regulated? reaction seems to generate a substantial population of memory How is GC B cell differentiation regulated? Is there is an actual phenotype B cells, which have been identified as early as day fate decision per se, or is differentiation the result of an accumu- 7 postimmunization (70); this observation led to the plausi- lation of factors that overrides a death or GC-retention signal? ble proposal that some early memory cells derive indepen- h ora fImmunology of Journal The Table I. Effects of genetic manipulations of key molecules in the humoral response on GCs, PCs, and Bmem cells

Humoral Phenotype Gene-Targeted Deficiency GC Formation and Th Cells Ab/AFCs/PCs Bmem Cells References CD40 No GC formation (measured by histology + PNA after No switched Ab to TD Ag (not TI) No secondary GC formation (10) secondary immunization) CD40L No GC formation (measured by histology + PNA) Primary TD, but not TI, IgM responses decreased. Secondary Secondary IgG1 absent (11) IgG1 absent. CD40L-block No GC formation if administered early. Later After adoptive transfer of primed B cells, IgG1 titers reduced (4, 7, 12) administration after immunization reduces GC after secondary immunization. BM AFCs were lower-affinity (measured by histology + PNA). if block occurred after GC formation but no effect on af- finity. ICOS Reduced or no GC formation (measured by histology + IgG1 decreased. Decreased switching. (13–16) PNA). Decreased IL-4. ICOSL No GC formation (measured by histology + PNA). IgG1 decreased. Decreased affinity. Secondary GCs decreased (17, 18) Decreased IL-4. ICOS-block Days 5, 6, 7 block: GC cells reduced. Days 5, 6, 7 block: PC number normal but high-affinity re- Days 5, 6, 7 block: Bmem cell number normal. (19) duced. Lower-affinity memory compartment. Sec- ondary responses of normal magnitude but lower affinity. SAP Decreased GC (measured by histology + PNA) Long-lived IgG Ab decreased Memory decreased (measured by ELISPOT) (20) LTa No GC formation (as measured by histology + PNA). Low doses NP-OVA: drastically reduced Ag-specific IgG1, (21) Perturbed FDC organization. SHM found. high doses IgG1 response comparable to WT. 2/2 CD80 and CD86 GCs form2 2 in CD80 but very little in Decreased Ab and AFCs when both absent (22) CD86 / (measured by histology + PNA) CD86 block Early (days 1–3) injection: number of mutations in GC Early (days 1–3) injection: Ab reduced by 50%, late (days 6– Early (days 1–3) injection: secondary responses (4) cells reduced. 10) modest effect. were decreased by measurement of Ab. CD28 No GC formation (measured by histology + PNA); B cells IgG titers reduced (23) formed clusters but did not undergo expansion nor acquire somatic mutations. PD ligands Capable of GC formation, but GCs declined late in the Long-lived PCs decreased, more high-affinity PCs. Bmem cell numbers were comparable to WT or (24) response. even increased PD-1 Capable of GC formation, but GCs declined late in the Long-lived PCs decreased, more high-affinity PCs. Bmem cell numbers were decreased (24) response. Increased TFH s but less IL-4 and IL-21. IL-4Ra Decreased GCs Decreased AFC and affinity of AFC. Class-switch was im- (25, 26) paired. IL-21 and IL-21R GCs formed but were not maintained (impaired Ab was decreased, and class-switch was impaired, Zotos et al. (28): more Bmem cells but of lower (27–29) proliferation). TFH s formed, but numbers were variable affinity. Linterman et al. (29): early memory at different time points. IL-21 acted directly on GC comparable to WT. B cells to maintain Bcl-6 expression. CD45 GCs formed but were not maintained Decreased Ab production (32) FAS Increased and sustained GC, which was CD40L Increased number of, and mutations/gene in, (33, 34) dependent. memory. B cell leukemia-xL Tg GC and IgG1+ Ab normal Lower-affinity BM AFC and Ab due to retention of low-affinity (6) cells rather than impaired formation of high affinity Bcl-2 Tg Increased number of GCs Increased numbers of AFCs; retention of low-affinity splenic Increased numbers of Bmem cells due to (35–37) AFCs; BM AFC affinity was similar to WT. retention of low-affinity memory cells BIM Increased number of GCs Increased numbers of AFC; retention of low-affinity splenic Increased numbers of Bmem cells due to (35) AFC; BM AFC affinity was similar to WT. retention of low-affinity memory cells BCL-6 (hematopoietic GC formation and TFH s were absent Produced normal levels of IgM and IgG1 until day 14. IgG1 Bmem cell numbers were normal but no SHM (38–40) chimera) levels decreased over time with few long-lived IgG1 AFCs. IgM remained strong in primary and secondary. Low- affinity IgG1 Ab produced in primary and secondary responses. Phospholipase Cg2 GCs reduced Reduced IgG1 AFCs after primary and secondary Bmem cells reduced (41) CD21/35 GCs reduced Long-lived Ab and AFCs decreased but higher-affinity cells (42–44) remained

CD19 hypomorph GCs reduced and failed to progress Loss of long-lived Ab Reduced secondary response (45) 3119

AFC, Ab-forming cell.

Downloaded from from Downloaded http://www.jimmunol.org/ by guest on September 23, 2021 23, September on guest by 3120 BRIEF REVIEWS: INFLUENCE OF GC DYNAMICS ON B CELL MEMORY dently of the GC (19). Similarly, short durations of BrdU mutant CD19 alleles form GCs, but these GCs fail to mature administration conducted early in the GC response, in which (45), and there are significant defects in the long-term Ab re- cells that stop dividing shortly after incorporating BrdU will sponse. Although it was originally thought that ICOS defi- remain BrdU+, lead to very efficient labeling of many long- ciency completely blocked the GC response (13–15, 17, 18), lived Bmem cells, as detected at week 8 and beyond postimmu- ICOS seems to be in the middle of these two categories. More nization (66) (S. Anderson and M. Shlomchik, unpublished detailed research showed that GCs do form in the absence of observations). Takahashi et al. (33) found that the memory this molecule, albeit to a much lesser degree; ICOS blockade compartment was much less mutated than the PC compart- induced during the GC response leads to a reduction in VH ment, and its level of mutation matched that of the early, but mutations in Bmem cells but not the total numbers of memory not late, GC population, again indicating that memory cells cells themselves (16, 19). Importantly, total Ab responses and emerge relatively early from the GC. Conversely, the frequency particularly high-affinity Ab were reduced by such treatment. of mutations among long-lived PCs in the bone marrow (BM) ICOS may act, in part, to sustain CD40L expression by T cells, matches that of the late, but not the early, GC (33). Together which, in turn, is required to maintain CD40 expression on with evidence that the size of the long-lived PC compartment in B cells (16). In the absence of signaling through receptors of the BM continues to increase (7) even beyond 16 wk postim- T cell-derived cytokines, GCs can form, but their frequency is munization, a time when the memory compartment has long decreased in the late response. In the case of IL-4, there was a 3- since stabilized (66), it seems that the late output of the GC is fold reduction in the frequency of GC B cells in mesenteric more skewed to the generation of PCs, some of which migrate lymph nodes 2 wk postinfection with an intestinal parasite Downloaded from to and reside in the BM as part of the long-lived PC pool. (25), whereas in lymph nodes responding to (4-hydroxy-3- Commensurate with this interpretation, the average affinity nitrophenyl)acetyl (NP)-OVA infection, there was a decrease of the BM PC population increases for 8–12 wk after immu- in the frequency of GC B cells in IL-4–deficient animals only nization with a nonreplicating Ag (7, 71). at days 14 and 21 (26). IL-21 acts directly on GC B cells to maintain Bcl-6 expression; without it, the GCs dissipate later

A variety of mutations disrupt the GC in the response (28, 29). Still other molecules are important in http://www.jimmunol.org/ Given the complex T–B interplay required to generate and then the maintenance of even later stages of the GC response, sug- sustain a GC response, it is not surprising that a large number gesting that constant B–T interactions are important for main- of mutations affecting molecules and cells critical to the im- tenance of GC, although the proteins responsible change as the mune system disrupt the GC reaction, to at least some degree. GC evolves. Our recent research demonstrated that the absence Although each of these mutations has its own distinct phe- of PD-1 resulted in increased apoptosis in the late GC, as well notype, it is interesting to consider them with respect to the as smaller GCs at these time points (24). Although the num- timing and nature of the defects with which they are associated. bers of cells of a TFH phenotype increased during the late GC

One category of such mutations blocks signals and/or cellular response in the absence of PD-1 signals, they produced less by guest on September 23, 2021 interactions critical for the formation of GCs or the very early IL-4 and IL-21 directly ex vivo. This correlated with a decrease events involved in GC development. Mice deficient in Bcl-6, in survival of GC B cells, similar to the IL-4Ra and IL-21 SAP, CD40, or CD40L do not form GCs (10–12, 20, 39) gene-targeted mice (24, 25, 28, 29). Therefore, B and T cells (Table I). Bmem cell numbers, as well as long-lived PCs and Ab interact to direct the formation of GC responses, as well as the titers, are also markedly diminished in these animals, consis- maintenance of the TFH phenotype (28, 29) and, as a result, tent with the lack of GC formation. Similarly, human patients the maintenance of the late GC response. with deficiencies in CD40, CD40L, ICOS, and SAP have Quality of Bmem cells and quantity and quality of PCs are determined dramatically decreased numbers of switched Bmem cells. In contrast, they often have normal or slightly reduced numbers in the late GC response + + of mutated IgM CD27 B cells, with CD27 used as a marker Although numbers of Bmem cells are not greatly reduced in the for human memory cells (8). In mice, low-affinity Bmem cells face of mutations that block GC progression, the affinity of the can be formed in the absence of Bcl-6 (39). Taken together, resultant memory and/or PC populations can be affected, these data suggest that early signaling events, which occur depending on the mutant (Table I). The process that controls before the formation of a functional GC, are sufficient to in- selective enrichment of high-affinity cells in the GC population duce a degree of Bmem cell formation (Table I). However, in and their subsequent recruitment into the long-lived popula- the absence of these signals, the numbers of Bmem cells are tions could be influenced by several factors. In the absence of nonetheless markedly reduced, presumably because a GC en- competition between cells of varying affinities, low-affinity GC vironment in which Bmem cell precursor expansion takes place B cells are intrinsically more prone to undergo apoptosis com- is absent. In all of the mutants in this category, isotype- pared with high-affinity B cells (73). The presence of compet- switched long-lived Ab responses were severely impaired, as ing high-affinity cells also suppresses the response of low- expected from the lack of a GC response. affinity cells, although the mechanisms underlying this are un- Another category of mutations is at least somewhat per- known (74). When the balance between pro- and antiapoptotic missive for GC formation but seems to block GC progression or molecules downstream of the BCR is artificially tipped toward maintenance. These mutants that affect late GC responses tend survival, such as in B cell leukemia-xL transgenic (Tg) and Bim- to show differential regulation of Bmem cells and PCs, with deficient mice, there is an accumulation of low-affinity generally normal or even increased numbers of memory cells Bmem cells (6, 35). In contrast, in Fas-deficient mice, B cells but decreased numbers of PCs (Table I). For example, phos- continue dividing within the GC, undergoing more rounds pholipase Cg2 (41) and CD21/35 (42, 72) are vital for GC of SHM than usually occur in normal mice, resulting in a B cell survival but not formation. Similarly, mice expressing more mutated Bmem cell population (33, 34). Administering The Journal of Immunology 3121

CD40L-blocking Ab in the late GC response suppresses this typically and functionally heterogeneous. The identification of accumulation, suggesting that extension of the GC response such diversity within the Bmem cell pool raises additional, in- itself was responsible for the alteration of the memory popula- teresting questions about the signals that influence Bmem cell tion, rather than a direct effect of the absence of Fas signaling in formation. How would variations within the memory compart- Bmem cells (33). ment arise? In fact, there are almost no data that speak to this The availability of T cell help provides further extrinsic question. However, we speculate that in much the same way that modulation of selection. In the absence of signals through stochastic interactions with environmental cues could dictate IL-21 (28, 29), there is an accumulation of low-affinity GC cell fates in general, diverse experiences and timing could Bmem cells. In contrast, in the absence of PD-1 (24) or com- result in variation of Bmem cell properties. Specifically, it is likely plement receptor 2 (43) signaling, the affinity of the Bmem cell that the specific nature of the components of the primary re- pool seems to be relatively unaffected, but the remaining long- sponse alter the types of Bmem cells produced. These alterations lived PCs are of higher affinity than the wild-type (WT) con- may include the type and amount of the infectious agent, route trols. In the case of PD-1 deficiency, this could be related to the of infection, number of precursor cells, and types of TH cells partial reduction of IL-4 and IL-21 found at late GC time (and thus, cytokines). points (24). Although the results from IL-21– and PD-1– deficient animals may seem contradictory, we hypothesize Subsets of Bmem cells that the difference can be explained by the differing degrees Defining subsets of Bmem cells requires the phenotypic, rather of T cell help available in the two systems (Fig. 1). In the than functional, identification of B cell memory, something Downloaded from complete absence of IL-21, low-affinity cells are produced in that until recently has mainly been pursued using cells from the initial phase of the response. As a result of the lack of IL-21 human tissues or blood. Studies of human Bmem cells have long signals, Bcl-6 expression is not maintained in GC B cells, and used CD27 as a marker of Bmem cells, although it subsequently 2 they exit from the GC too early to produce substantial num- was found that there is a small subset of CD27 , isotype- bers of high-affinity cells. As a result, low-affinity post-GC switched B cells (77–79). The notion that CD27 marked hu- http://www.jimmunol.org/ (memory phenotype) cells accumulate; in fact, there is an ex- man Bmem cells derived initially from the finding that this cess of such cells (28). This could reflect more that the process subset contained the vast majority of isotype-switched and was terminated prematurely rather than a direct effect on se- somatically mutated B cells, which, strictly speaking, only indi- lection per se. In contrast, in the absence of PD-1 signaling, cates that the cells have undergone prior activation. IL-4 and IL-21 production was reduced, but not absent; this Heterogeneity of BCR isotype is the best-studied aspect was most evident at later time points (24). When low- and of diversity within the memory compartment. In humans, high-affinity cells are formed, but there is reduced T cell help, CD27+ B cells can be subdivided into isotype-switched and + + + competition for T cell help could result in high-affinity cells nonswitched (IgM )Bmem cells. However, these IgM CD27 outcompeting the low-affinity cells. This idea is supported by cells could be found in patients with CD40L deficiency, sug- by guest on September 23, 2021 data showing that when competition with high-affinity cells is gesting to some that they were not real memory cells, because reduced, even very low-affinity cells are capable of forming they could form in the presumed absence of a functional GC GCs and entering the Bmem cell pool (75). Because PCs are (8). Yet, Bmem cells can be generated in mice in which a fully the main output of the late GC reaction, the net result is seen functional GC does not form (Table I). Therefore, these B cells within the PC compartment, which is smaller and particularly are formed independently of a GC, or signaling that takes place lacking in lower-affinity PCs when PD-1 signals do not oc- during early GC formation is enough to produce some Bmem cur (24). Therefore, it is possible that the levels of cytokines, cells before abrogation of the reaction occurs. There are and possibly other undefined T cell-derived signals, can affect other indications from murine studies that GC-independent the quality of the long-lived populations by affecting selection Bmem cells can be formed. Responses to TI Ags can form mem- of high-affinity GC precursors. ory (80), but whether these cells begin to undergo a TI GC Integrating these findings, we propose a model for how a pathway is unclear. Unmutated, low-affinity memory cells population of Bmem cells is formed. After each division, an ac- are found in immune mice with B cells lacking Bcl-6 and, tivated B cell can continue dividing, or it can exit the cell cycle hence, lacking GCs (39). Similarly, B1b cells can generate TI and differentiate into a memory cell or PC. This can occur within IgM memory cells that protect from Borellia infection (81). or outside of a GC. However, this fate decision is regulated in It is quite likely that, in addition to isotype, TI and TD a stochastic fashion by the microenvironment, with some signals Bmem cells differ qualitatively with respect to function and [e.g., those that downregulate Pax5 (76)] favoring PC over maintenance; indeed, NP-specific Bmem cells generated memory cell commitment, or vice versa. Within the GC, cells with TI immunization have a half-life similar to naive B cells expand and mutate; although low- and high-affinity cells may (75), in contrast to the very extended half-life of TD NP- differentiate into Bmem cells, competition for T cell help favors specific Bmem cells (66). survival of the fittest, presumably the high-affinity cells (Fig. 1). To extend the debate about the origin of IgM+CD27+ B cells Therefore, T cells and their products influence the magnitude in humans, researchers recently transferred human B cell subsets and quality of the response, as well as the ratio of Bmem cells/ into immunodeficient mice and immunized them with TI or TD PCs, in part depending on when during the GC response in- Ag (82). After immunization with the TD Ag, IgM+CD27+ and dividual mediators and mutations exert their effects (Fig. 1). isotype-switched CD27+ B cells responded by making IgG Ab, indicating a memory-like function. Other studies of human Heterogeneity within the Bmem cell pool IgM+CD27+ B cells showed somatic mutations in bcl6,anevent Although, in general, B cell memory has been referred to in that occurs in the GC (83), and the presence of V-region muta- + + monolithic terms, the Bmem cell population is actually pheno- tions in many IgM CD27 B cells suggests that they are bona fide 3122 BRIEF REVIEWS: INFLUENCE OF GC DYNAMICS ON B CELL MEMORY Downloaded from

FIGURE 1. Differential effects on long-lived B cell populations by IL-21 or PD-1. A, Model of the formation of quality and quantity of memory cells and PCs http://www.jimmunol.org/ + + during regulation of GC cells (blue) and the role of IL-4 TFHs (red) and IL-21 TFHs (green). TFHs that secrete cytokines other than IL-4 or IL-21 (such as IFN-g)orTFHs that do not secrete optimal amounts of cytokines are gray. During the GC response, cytokines secreted by TFHs promote GC cells to undergo survival, proliferation, and isotype switching and play a role in selection. During the early GC response, many low-affinity Bmem cells are formed. As the GC progresses, a combination of competition between high- and low-affinity cells for TFH-derived signals and the increased tendency of low-affinity cells to undergo apoptosis tends to enrich for high-affinity cells in the memory, although affinity will remain variable. High-affinity PCs are mainly generated during the late GC response. Phenotypes of mice deficient in IL-21 or IL-21R (B) or PD-1 (C). In the absence of these molecules, the early GC response is comparable to WT (A–C). B, However, in the absence of IL-21, Bcl-6 expression in GC B cells is not maintained at the height of the response, leading to a decrease in GC cell number and PCs. As a result, it is likely that GC cells do not survive long enough to produce high-affinity cells, or high-affinity GC cells do not undergo expansion. Therefore, the long-lived populations remain relatively low affinity. C, In the absence of PD-1, the memory population is formed normally; however, in the late response, by guest on September 23, 2021 GCs are not maintained as a result of defective cytokine secretion from TFHs and, thus, the PC pool is reduced. Correspondingly, there is an increase in TFHs that produce less or no IL-4 and IL-21 (gray). High-affinity GC cells may outcompete low-affinity cells for the reduced levels of survival signals, surviving to become PC precursors in the late GC response. For this reason, although the PC pool is decreased, it consists of more high-affinity PCs than WT (A). memory cells. The presence of mutated IgM memory cells in memory cells with different (IgG, IgA, and IgE) isotypes. The mice has long been known (84), but these cells are understudied. importance of producing different isotypes lies in the ability of We showed that such cells are common in immune mice that the immune system to effectively respond appropriately to received small numbers of NP-specific B cells with the B1-8 a multitude of foreign Ags and at different anatomical sites, such knockin H chain; such IgM memory cells retained some depen- as by producing IgA in mucosal tissues. Interestingly, studies of dence on B lymphocyte stimulator (BLyS) for survival, unlike human cells show that multiple subsets of TFHs, as defined by their IgG1-expressing counterparts, indicating functional hetero- differential production of cytokines, can be induced in the geneity (85). Recently, the existence of murine IgM memory cells presence of the same cytokine milieu (87). Furthermore, iso- was confirmed by use of an AID-Cre YFP-reporter mouse, which lation and study of B cell–T cell conjugates demonstrated that demonstrated that IgM and switched Ag-experienced B cells are the same response could induce TFH subsets, as defined by formed in response to immunization with sheep RBCs or NP- distinct patterns of cytokine production, which, in turn in- chicken gamma globulin (71). Transfer of IgM+YFP+ (putative duced B cells with diverse, corresponding isotypes (26). Al- memory) cells induced GC formation in a secondary response, though more work is required to explore the presence, timing, + whereas transfer of isotype-switched YFP cells induced a pre- and location of TFH subsets during an immune response, it is dominantly plasmablast response, leading the investigators to possible that they play a role in producing diversity of the conclude that the heterogeneity seen in Ig isotypes directly relates Bmem cell pool, inducing switching of most B cells to one to different functions during a secondary response. Whether this isotype, as well as producing some B cells of other isotypes, was purely a correlative finding or whether the isotype differences allowing for plasticity during the secondary response. were mechanistically responsible for the different responses of Phenotypic differences within the memory compartment IgM and IgG Bmem cells was not determined; however, differ- ences in IgM and IgG1 cytoplasmic tails were hypothesized to Comparisons of human naive, IgM, and switched Bmem cells account for the rapid differentiation into Ab-forming cells by demonstrated that IgM and switched Bmem cells are genotypi- Bmem cells (86). cally and phenotypically similar and behave similarly in culture In addition to differences between switched and unswitched to various stimuli (56). Although they express many of the same isotypes, further heterogeneity lies in functional differences of proteins, the abundance of some of these proteins does differ, The Journal of Immunology 3123 suggesting slightly different experiences during the primary re- In addition to parameters that affect GC dynamics, other sponse and potentially underlying subtle differences in in vivo fine-tuning mechanisms, such as selection of affinity variants responses (56). Unfortunately, studying variations in human and control of Bmem cell numbers postexit of the cell cycle, Bmem cells is limited because comparisons between cells gener- exist to shape the Bmem cell repertoire. These processes all ated by the same Ag cannot be performed. However, differences converge to form an optimized, yet phenotypically and func- have also been found in murine Bmem cells generated in response tionally diverse, Bmem cell population. to the same Ag. Recent work from our laboratory showed that certain surface markers are differentially expressed on Bmem cells. Conclusions Subsets of Bmem cells differentially upregulate CD80, PD-L2, To survive, an organism must be able to respond to multiple CD73, and CD21/35 (59, 88 and M. Tomayko, N. Steinel, pathogens and appropriately adapt to variations in Ag stimu- S. Anderson, and M. Shlomchik, manuscript in preparation). lation and availability and type of T cell help. Therefore, These markers are induced independently of each other, again heterogeneity in the Bmem cell pool would be adaptive with consistent with a model that stochastic events, rather than de- respect to the variety of different infectious agents, recurring terministic signals, influence their expression. Nonetheless, there infections, and/or evolution of viruses over time. Furthermore, is a hierarchy, such that some memory cells express multiple to provide protection over the lifetime of the host, the memory-specific markers and others express few or none. These Bmem cell pool must also persist in the face of what could be subsets have differential rates of mutation, as well as BLyS de- many antigenic challenges. Diversity that includes some cells pendency, suggesting a “maturation gradient,” with the cells with more self-renewal capacity or ability to re-enter the GC Downloaded from lacking expression of these markers having fewer mutations reaction and others with a greater tendency to quickly differ- and greater BLyS dependency, more like naive B cells, whereas entiate into plasmablasts would be one way to ensure this. The the cells expressing multiple markers (particularly PD-L2 and next phase of investigation into Bmem cell development may CD80) seem to have more classical, fully-developed memory- include testing of whether different infections, through differ- like features (M. Tomayko et al., manuscript in preparation). In ent dynamics of the response and/or different innate immune addition to IgM Bmem cells, Dogan et al. (71) divided Bmem cells signals, affect the make-up of the Bmem cell pool. If so, it may http://www.jimmunol.org/ 2 into peanut agglutinin (PNA)+ and PNA fractions; however, help to explain why some vaccinations and infections induce these subsets were only present in a chronic response (71). memory over the lifetime of the host, whereas others do not. Therefore, it is possible that the PNA+ fraction had only recently exited the GC or may have even contained persistent GC cells; Acknowledgments thus, it may not form part of the memory response in the absence We thank Mary Tomayko, Steven Kerfoot, Saheli Sadanand, and Griselda of Ag. Nevertheless, this article clearly confirmed the existence of Zuccarino-Catania for helpful discussion and/or critical review of this manu- 2 IgM memory cells that were PNA . Lastly, identification of a script. +

subset of FCRL4 tissue-resident memory cells in humans (9, by guest on September 23, 2021 89) suggests that location may also play a role in producing spe- Disclosures The authors have no financial conflicts of interest. cialized subsets of Bmem cells. Overall, there is an emerging lit- erature that is uncovering substantial diversity in murine and human Bmem cells, not just at the level of the BCR isotype, but References also with respect to expression of key surface markers, although 1. Ahmed, R., and D. Gray. 1996. Immunological memory and protective immunity: understanding their relation. Science 272: 54–60. much more needs to be learned about the functional significance 2. Toellner, K. M., W. E. Jenkinson, D. R. Taylor, M. Khan, D. M. Sze, of these newly defined subsets. D. M. Sansom, C. G. Vinuesa, and I. C. MacLennan. 2002. Low-level hyper- mutation in T cell-independent germinal centers compared with high mutation rates associated with T cell-dependent germinal centers. J. Exp. Med. 195: 383–389. 3. de Vinuesa, C. G., M. C. Cook, J. Ball, M. Drew, Y. Sunners, M. Cascalho, M. Wabl, G. G. Klaus, and I. C. MacLennan. 2000. Germinal centers without Fine-tuning of the primary response results in a Bmem cell gradient T cells. J. Exp. Med. 191: 485–494. We propose that the quality of the Bmem cell population 4. Han, S., K. Hathcock, B. Zheng, T. B. Kepler, R. Hodes, and G. Kelsoe. 1995. Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in estab- depends on the time spent within the GC, as well as the quality lished germinal centers. J. Immunol. 155: 556–567. of interactions with T cells while there. This results in a gra- 5. Jacob, J., G. Kelsoe, K. Rajewsky, and U. Weiss. 1991. Intraclonal generation of antibody mutants in germinal centres. Nature 354: 389–392. dient of Bmem cell maturation, with the more classically defined 6. Takahashi, Y., D. M. Cerasoli, J. M. Dal Porto, M. Shimoda, R. Freund, W. Fang, Bmem cells emerging later in the response, although before D. G. Telander, E. N. Malvey, D. L. Mueller, T. W. Behrens, and G. Kelsoe. 1999. most of the PCs. Within this gradient, one could broadly Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice. J. Exp. Med. 190: 399–410. characterize some Bmem cells as more naive-like, and others as 7. Takahashi, Y., P. R. Dutta, D. M. Cerasoli, and G. Kelsoe. 1998. In situ studies of more (classically defined) memory-like. Hence, we would pre- the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. V. Affinity mat- uration develops in two stages of clonal selection. J. Exp. Med. 187: 885–895. dict that TI and early Bmem cells, as well as Bmem cells from 8. Tangye, S. G., and K. L. Good. 2007. Human IgM+CD27+ B cells: memory B cells mutants that block early GC progression, are more naive-like or “memory” B cells? J. Immunol. 179: 13–19. 9. Tangye, S. G., and D. M. Tarlinton. 2009. Memory B cells: effectors of long-lived and less mutated, with reduced expression of memory-specific immune responses. Eur. J. Immunol. 39: 2065–2075. surface markers. Such cells may be more likely to re-enter GCs 10. Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, upon Ag re-exposure. More memory-like cells would have re- N. Yoshida, T. Kishimoto, and H. Kikutani. 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal cen- ciprocal properties and would tend to form only in TD ter formation. Immunity 1: 167–178. responses, perhaps as a consequence of more receipt of T cell 11. Xu, J., T. M. Foy, J. D. Laman, E. A. Elliott, J. J. Dunn, T. J. Waldschmidt, J. Elsemore, R. J. Noelle, and R. A. Flavell. 1994. Mice deficient for the CD40 signaling and/or numbers of division or other signals received ligand. [Published erratum appears in 1994 Immunity 1994 Oct;1(7):following in the GC. Therefore, the heterogeneity of the Bmem cell 613.] Immunity 1: 423–431. 12. Foy, T. M., J. D. Laman, J. A. Ledbetter, A. Aruffo, E. Claassen, and R. J. Noelle. population is a result of fine-tuning mechanisms during the 1994. gp39-CD40 interactions are essential for germinal center formation and the primary response. development of B cell memory. J. Exp. Med. 180: 157–163. 3124 BRIEF REVIEWS: INFLUENCE OF GC DYNAMICS ON B CELL MEMORY

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OCTOBER 8-9, 2010

THE UNIVERSITY OF CHICAGO 924 East 57th Street Chicago, Illinois

KEYNOTE SPEAKER: IRUN COHEN, WEIZMANN INSTITUTE SPEAKERS: John Cambier, National Jewish Medical and Laurence Morel, University of Florida Downloaded from Research Center Sylviane Muller, CNRS, Strasbourg Steve Clarke, University of North Carolina David Nemazee, Scripps Research Institute Keith Elkon, University of Washington Michel Nussenzweig, Rockefeller University Loren Erikson, University of Virginia Paul Plotz, National Institute of Health Haochu Huang, University of Chicago Bill Robinson, Stanford University Greg Lemke, Salk Institute Ann Rothstein, University Mass Medical School Cliff Lowell, University of California, Eng Tan, Scripps Research Institute San Francisco Takeshi Tsubata, Tokyo Medical and Dental Center

Marc Monestier, Temple University PJ Utz, Stanford University http://www.jimmunol.org/ School of Medicine Thomas Winkler, University of Erlangen

Organizing Committee: Marko Radic, Mark Shlomchik, Tammy Utset, and Martin Weigert

Above: Autoantibody binding to NETs. For more information, please visit: http://knappcenter.uchicago.edu or Image courtesy of Dwivedi and Radic, UTHSC. email Angela Hayes [email protected] by guest on September 23, 2021

Get a GRIP: An AAI program designed to help new investigators prepare their NIH grant proposals AAI is pleased to offer a program to match new PI’s with established PI’s who have significant, successful grant writing careers. The Grant Review for Immunologists Program (GRIP) invites new PI’s to submit an outline or NIH-style abstract to the GRIP coordinator who, with the assistance of a small volunteer subcommittee, will attempt to match the topic of the proposal with the research experience of an established PI. Matches will be made as quickly as possible to allow new PI’s to meet upcoming NIH grant deadlines. Participation is strictly voluntary and is not intended to supplant internal mentoring programs. GRIP is now accepting both new PI and established PI participants. Please send your CV and a brief description of either your potential research project (new PI’s) or grant reviewing experience (established PI’s) to [email protected] (please write “GRIP” in the subject line). Program details at www.aai.org/GRIP_rd.htm