(2016) 30, 2283–2292 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0887-6924/16 www.nature.com/leu

REVIEW Human memory B cells

M Seifert and R Küppers

A key feature of the adaptive is the generation of memory B and T cells and long-lived plasma cells, providing protective against recurring infectious agents. Memory B cells are generated in (GC) reactions in the course of -dependent immune responses and are distinguished from naive B cells by an increased lifespan, faster and stronger response to stimulation and expression of somatically mutated and affinity matured immunoglobulin (Ig) genes. Approximately 40% of human B cells in adults are memory B cells, and several subsets were identified. Besides IgG+ and IgA+ memory B cells, ∼ 50% of peripheral memory B cells express IgM with or without IgD. Further smaller subpopulations have additionally been described. These various subsets share typical memory features, but likely also fulfill distinct functions. IgM memory B cells appear to have the propensity for refined adaptation upon restimulation in additional GC reactions, whereas reactivated IgG B cells rather differentiate directly into plasma cells. The human memory B-cell pool is characterized by (sometimes amazingly large) clonal expansions, often showing extensive intraclonal IgV gene diversity. Moreover, memory B-cell clones are frequently composed of members of various subsets, showing that from a single GC B-cell a variety of memory B cells with distinct functions is generated. Thus, the human memory B-cell compartment is highly diverse and flexible. Several B-cell malignancies display features suggesting a derivation from memory B cells. This includes a subset of chronic lymphocytic leukemia, and . The exposure of memory B cells to oncogenic events during their generation in the GC, the longevity of these B cells and the ease to activate them may be key determinants for their .

Leukemia (2016) 30, 2283–2292; doi:10.1038/leu.2016.226

INTRODUCTION GENERATION OF B CELLS The success of vaccination in eradicating life-threatening infec- Throughout life, B cells are generated from lymphoid precursors. tions is often considered as the most important medical break- B-cell generation occurs in the marrow in a tightly regulated through in the past century. The that is process, with stepwise recombination of V, (D) and J gene generated upon vaccination rests on our segments coding for the variable (V) region of the immunoglo- 1 and its two key cell types, B and T . Although innate bulin (Ig) heavy and light chains. If a B cell succeeds in producing immunity can cope with most infectious insults, the severe a functional, non-autoreactive BCR, it differentiates into a mature, diseases that result from deficiencies in the function of B and/or . These cells express the BCR as IgM and IgD molecules, recirculate through the body and account for ∼ 50% of B cells in T cells demonstrate the important role of the adaptive immune 2 system. The key to adaptive immunity is the expression of highly adults. As multiple V, D and J gene segments are available for V diverse receptors on T and B lymphocytes, the T-cell (D)J recombination, and as additional variability is generated at the joining sites, each newly generated B cell is equipped with a and the B-cell receptor (BCR), respectively, that enable a unique BCR that hence represents a clonal marker. In this way, a highly specific response against all types of foreign . highly diverse primary repertoire of naive B cells is generated.1 A further hallmark of the adaptive immune system is the generation of immune memory. This is provided by memory lymphocytes that are generated in immune responses against GENERATION OF MEMORY B CELLS IN THE GERMINAL CENTER specific antigens, are long-lived and provide an enhanced and REACTION improved immune reaction upon reencounter of cognate antigen. Upon strong BCR stimulation, B cells can proliferate and Thereby, memory lymphocytes protect the organism from differentiate into plasma cells (PCs) without the need for T-cell diseases caused by reinfection with the same or similar infectious support. Such T cell-independent (TI) immune responses generate agents, sometimes for decades. PCs producing of low affinity and typically do not give In recent years, considerable progress has been made in our rise to memory B cells.3 If an antigen can be presented to T helper understanding of the generation and diversity of memory B cells, (Th) cells via major histocompatibility complex class II molecules, a their distinct subsets and functions. However, several aspects of T cell-dependent (TD) humoral is initiated. this topic are still unresolved or debated. We present here an B cells are activated in the periphery and migrate into secondary overview on human memory B cells, addressing these issues and lymphoid tissues, or are activated locally by floating antigen or we also consider the pathogenesis of -derived immune complexes, presented by dendritic cells. The activated B malignancies. cells meet antigen-specific Th cells at the border of the T-cell zone

Institute of Cell Biology ( Research), Medical Faculty, University of Duisburg-Essen, Essen, Germany. Correspondence: Professor R Küppers, Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Virchowstraße 173, 45122 Essen, Germany. E-mail: [email protected] Received 24 May 2016; revised 29 June 2016; accepted 26 July 2016; accepted article preview online 8 August 2016; advance online publication, 26 August 2016 Human memory B cells M Seifert and R Küppers 2284 and B cell follicle.4 The cognate B-cell–Th cell interaction leads to a undergoes class-switch recombination. In this process, the further stimulation of both types of cells in primary focus originally expressed Cμ and Cδ genes are replaced by one of reactions, where they proliferate, and short-lived, mostly IgM the 3′ located CH genes, Cγ1-4, Cα1 and 2 or Cε. These encode secreting, PCs are generated.4 heavy chains that equip secreted antibodies with distinct effector A fraction of the activated lymphocytes migrates into B-cell functions.1 follicles and seeds germinal center (GC) reactions.1,5 These GC Induction of the PC program is regulated by the transcription founder B cells upregulate BCL6, the master regulator of the factors PRDM1, IRF4 and XBP1.10 PRDM1 and IRF4 both suppress GC program, already before GC entry and vigorously proliferate BCL6, and thereby the GC B-cell program, and induce together (Figure 1).6 Proliferation in the GC is accompanied by somatic with XBP1 the expression of PC-specific genes.10 Notably, class hypermutation (SHM) that introduces mutations at a rate switching can also bias GC B cells toward PC differentiation.11 In − − of 10 3–10 4/bp/cell division into the rearranged IgV genes. contrast, the regulation of memory B-cell differentiation is poorly SHM requires strong transcriptional activity of the IgV genes.7 SHM understood. The transcription factor nuclear factor-κB, which is not only targets the IgV genes, but with lower frequency also induced in by CD40 and BCR triggering and can some non-Ig genes transcribed in GC B cells, including BCL6.8 downregulate BCL6, is involved in this process,12 and - Despite some preferences, somatic mutations occur in principle 24 can also enhance memory B-cell formation.13 In mice, the randomly downstream from the IgV promoter over a region of transcription factor Bach2 plays a key role in selecting GC B cells ∼ 1 kb. Most mutations will cause an amino acid exchange.7 The with intermediate affinity into the memory B-cell compartment.14 vast majority of such replacement mutations will be disadvanta- Plasmablasts may migrate into specialized niches in the bone geous, for example, if reducing BCR affinity. Hence, most marrow, where they terminally differentiate into long-lived PCs, progenies of a somatically mutating B-cell clone represent secreting large amounts of high-affinity over extended unfavorable variants that are eliminated by apoptosis.1 Only if a time periods.10 Memory B cells circulate through the body as BCR with improved affinity is expressed, the respective cell is resting lymphocytes until reactivation. Whereas the established positively selected through cognate interaction between GC high-affinity antibody titer in and blood represents a direct B cells and GC Th cells.9 This selection takes place in the light and continuously ongoing defense mechanism provided by PCs, zone of the GC, where GC B cells show little proliferative activity memory B cells require restimulation to provide enhanced and (centrocytes), whereas mutating and proliferating GC B cells improved immune responses. () are mostly restricted to the dark zone. GC B cells A vast majority of memory B cells are generated in GC reactions typically acquire one or a few mutations before migrating to the (Figure 1). However, in the mouse, some memory B cells are light zone. Positively selected centrocytes either return to the dark generated upon T-cell stimulation already before GC passage. zone for further proliferation and mutation or differentiate into These cells are partly class switched, carry unmutated IgV PCs or memory B cells.5 In the light zone, a fraction of GC B cells genes15,16 and likely represent progeny of the primary focus

Figure 1. Memory B-cell generation in the GC. (Left) After initial encounter of antigen-specific B and T cells at the border of T-cell zone and B-cell follicle (primary focus reaction), activated B and T cells migrate into the follicles and initiate GC reactions. Some human IgM+IgD+ B cells with features of memory B cells (CD23−ABCB1−) but scarcely mutated IgV genes are detectable. These might represent very early descendants of GC reactions or memory B cells generated at a pre-GC stage. (Middle) During early phases of GC reactions, preferentially IgM+IgD+ and only few IgM-only or class-switched memory B cells develop. (Right) During late stages of GC reactions, most GC B cells (and thus memory B cells generated from them) are class switched.

Leukemia (2016) 2283 – 2292 © 2016 Macmillan Publishers Limited, part of Springer Nature. Human memory B cells M Seifert and R Küppers 2285 reaction. Notably, only few human memory B cells lack somatic easily identified and isolated. However, the detection of somati- mutations, and these cells may have differentiated from early GC cally mutated IgM+ B cells pointed to the existence of IgM+ B cells before onset of SHM. Hence, it remains unclear whether any memory B cells.2,17,28,31 In humans, the tumor necrosis factor human memory B cells are generated before GC reactions receptor superfamily member CD27 became an important marker (Figure 1). to distinguish naive from mutated IgM+ B cells (note that GC B cells and post-GC PCs are also CD27+).2,36 It is essential to reveal specific generation pathways and distinct immunological func- CHARACTERISTICS AND FUNCTIONS OF MEMORY B CELLS tions of individual memory B-cell subsets to understand the Memory B cells differ in several key characteristics from naive B memory B-cell system. cells (Table 1). First, many memory B cells have undergone class switching. Second, their vast majority harbors mutated IgV IgG memory B cells 2,17 genes. Third, the BCRs of naive B cells typically show low Approximately 15–20% of peripheral blood (PB) B cells in adults affinity to the immunizing antigen, but memory B cells have + fi fi are IgG B cells, expressing mainly IgG1, IgG2 or IgG3, whereas gained intermediate to high af nity, as a consequence of af nity IgG4 is very rare.37 Their proportions among memory B cells vary maturation in the GC.1 Fourth, naive B cells have a lifespan of few 18 considerably among individuals, likely because of different weeks, whereas memory B cell clones can survive for fi 19,20 infection histories, as switching to speci c IgG subclasses is decades. Fifth, memory B cells proliferate and/or differentiate influenced by the type of antigen and various immunoregulatory faster and more efficient than naive B cells upon antigen-specific 21,22 factors. The existence of IgG subclasses contributes to the or polyclonal stimulation. This is because of several reasons, versatility of because of specific effector including a reduced expression of quiescence factors (KLF4, KLF9 functions. Approximately 20–25% of IgG memory B cells lack CD27 and PLZF), and higher expression/induction of costimulatory expression.37 The increase of this subpopulation in the elderly has molecules (SLAM, tumor necrosis factor, TLR7/9, CD27, CD80, CD86 led to the idea that these cells are aged or exhausted memory B and interleukin-21 receptor), antiapoptotic molecules (BCL2 family cells.38 IgG+CD27 − B cells are considerably less mutated and are members) and signal transducers (signal transducer and activator more frequently IgG3+ and less frequently IgG2+.27,39 However, 23–25 of transcription 3) by memory B cells. In addition, molecular both types of memory B cells often derive from common GC B-cell features of the class-switched BCR sustain memory B-cell clones.27,39 The specific characteristics of the two IgG+ memory 26 activation potentials. Finally, memory B cells are often clonally B-cell subsets indicate their common generation, but different 27–29 expanded and localized in areas of facilitated antigen kinetics or dependency on different microenvironmental encounter (for example, the marginal zone, Peyer’s patch dome stimulations. 30 region). IgG2 antibodies are the main type of IgG that is produced in TI When memory B cells are reactivated, they proliferate and immune responses. However, the vast majority of IgG2+ B cells 31–34 either differentiate into PC or reenter GC reactions. Newly show mutation levels similar to other IgG memory B cells,27,39 formed PCs quickly contribute large amounts of high-affinity arguing that IgG2+ B cells are also post-GC B cells. Thus, it appears antibodies to the serum pool. This increase is an essential function that class switching to IgG2 on one hand occurs during TI immune of memory B cells, as long-lived PCs lack proliferative potential responses that give rise to short-lived PCs and, on the other hand, and cannot supply an additional boost of high-affinity in TD immune responses in the GC that produce IgG2 memory antibodies.35 If reactived memory B cells reenter GC reactions, B cells. they may further adapt to modified pathogens or infection A peculiar subset of IgG+ B cells are IgG3+CD27− cells, because conditions by downstream class switching and/or new rounds of ∼ 20% of these cells are IgV gene unmutated and display the affinity maturation (Figure 2a).31 In these ways, memory B cells lowest mutation load among all IgG+ B cells.27 This may indicate improve immunity by adjusting to concentration, localization and that a fraction of these cells are derived from TI immune responses variability of infectious agents, and hence represent the adaptive or are generated in TD responses before GC entry. However, as a arm of immune memory. fraction of these cells carry BCL6 mutations (see below) and as many IgG3+ B cells are clonally related to conventional memory B cells,27 at least a large fraction of the IgG3+CD27− cells are GC HUMAN MEMORY B-CELL SUBSETS derived. Initial studies on memory B cells in mice and humans were In mice and humans, IgG memory B cells have the propensity to focused in particular on IgG+ memory B cells, as these can be differentiate into PCs upon reactivation, and mostly not to reenter

Table 1. Features of the main human mature B-cell subsets

Feature Naive B cells Mature GC B cells IgM+IgD+ IgM-only IgG+ and IgA+ CD5+ B cells CD27+ B cells B cells B cells

Approximate frequency in PB 50% 5% NA 15% 5% 25% Ig expression IgM and IgD IgM and IgD IgM or IgG IgM and IgD IgM IgG or IgA CD27 expression −−a + + + + (80%) Phenotypic markers CD23+, ABCB1+ CD5+ CD20high, CD38+ CD23− ABCB1− CD23− ABCB1− CD23− ABCB1− Mutated IgV genes −−/+a + +++ Longevity Weeks Weeks Short-lived Long-lived Long-lived Long-lived Propensity for differentiation Low High High Moderate Moderate High Ease of activation by BCR crosslinking Low High High High High High Presence at birth Yes Yes Few Few No No Presence in XHIGM patients Yes Yes No Yesb No No Abbreviations: BCR, B-cell receptor; GC, germinal center; Ig, immunoglobulin; NA, not applicable; PB, peripheral blood; XHIGM, X-linked hyper- . aApproximately 10% of mature CD5+ B cells express CD27 and carry somatically mutated IgV genes. bOnly at low frequency and with low IgV gene mutation frequency.

© 2016 Macmillan Publishers Limited, part of Springer Nature. Leukemia (2016) 2283 – 2292 Human memory B cells M Seifert and R Küppers 2286

Figure 2. Genealogical analysis of memory B-cell generation. Genealogical analysis of IgVH gene sequences from PB memory B cells. Typical results are shown, data taken from Budeus et al.27 Dendrograms begin with the germline configuration, lines between nodes represent single mutation events, colored nodes represent at least one and sometimes several clone members and cell types are according to color in the legend. Three main dendrogram types are detectable. (a) Mutated IgM+ or class-switched memory B cells may reenter GC reactions and give rise to memory B cells with typically few members with many shared mutations (deriving from the IgV mutated founder cell) and many individual mutations, indicative of intense competition during the secondary GC reaction. (b) Dendrograms based on IgM+IgD+CD27+ or IgM- only memory B cells alone show an early and broad diversification (bushy dendrograms). (c) Some dendrograms consist of multiple types of memory B cells, suggesting that single GC reactions can lead to highly diversified memory B-cell clones.

GC reactions.31–33 This propensity is linked to intrinsic signaling IgA memory B cells features of the IgG BCR and to low expression of the PC IgA memory B cells account for ∼ 10% of B cells in the PB and differentiation-inhibiting factor BACH2 by IgG memory B mostly express CD27. They are preferentially generated in immune cells.33,40,41 responses in the intestine and other mucosa-associated lymphatic

Leukemia (2016) 2283 – 2292 © 2016 Macmillan Publishers Limited, part of Springer Nature. Human memory B cells M Seifert and R Küppers 2287 tissues, and predominantly localized there.42 Indeed, IgA memory memory, highly similar to IgG+ memory, and significantly different B cells (and IgA PC) in the mouse express surface molecules that to naive B cells.22,33 Fifth, the presence of BCL6 mutations in 20% are associated with mucosal tissue homing.43 Although most IgA+ of IgM+IgD+CD27+ and 30% of IgG memory B cells28 strongly memory B cells are generated in TD immune responses, some PB argues for a GC derivation of all or at least the vast majority of IgA+ B cells lack CD27 expression, undergo less proliferation, show these cells, because the essential SHM factor activation-induced slightly lower IgV mutation levels than IgA+CD27+ B cells and, cytidine deaminase (AID) and its target BCL6 are only coexpressed thus, have been proposed to be generated independently of GC.44 in GC B cells5 and transcription of a gene is mandatory for SHM.7 Interestingly, these IgA+CD27− B cells express more frequently The, on average, one-third lower BCL6 mutation frequency among polyreactive antibodies with antibacterial specificity than IgA+ IgM+IgD+CD27+ B cells thereby reflects their one-third lower IgV CD27+ B cells.45 Of note, short proliferation history, low mutation gene mutation load. Sixth, the high frequency of clonally related level or polyreactivity do not irrevocably constitute a GC- IgM+IgD+CD27+ and post-GC memory B cells with both shared independent generation of IgA+ B cells, but may reflect a shorter and distinct IgV gene mutations strongly argues for a common GC GC participation. derivation (Figure 2b).27 Seventh, antigen-specific IgM+ memory B cells are detectable after various TD infections or in 62–64 IgE memory B cells humans. In light of the strong arguments for a GC derivation of at least a IgE-expressing memory B cells are hardly detectable in the PB of large fraction of IgM+IgD+CD27+ B cells, the question arises of healthy humans. It has been proposed that IgE class switching in whether the seemingly opposing views on the origin of these cells mice occurs mainly in some IgG1+ GC B cells that quickly can be resolved. Regarding repertoire peculiarities of IgM+IgD+ differentiate into PCs upon IgE switching, and that there are hardly CD27+ B cells, neither a unique IgV gene junctional diversity54 nor any IgE memory B cells.46 Indeed, further mouse models distinct IgV gene usage29 of IgM+IgD+CD27+ B cells was confirmed suggested a highly restricted lifespan of IgE memory B cells.47 in a comprehensive analysis.27 The early diversified BCR repertoire However, this view has been challenged by studies showing a of IgM+IgD+CD27+ B cells in young children may simply reflect functional murine IgE memory B-cell subset.48 Despite their that GC reactions in infants are less effective and produce class- central importance in asthma and allergic diseases, the genera- switched progeny less frequently, so that the first GC-derived tion, diversity or clonal interrelation of human IgE memory B cells memory B cells consist of a more diverse IgM than IgG memory are poorly understood. B-cell repertoire (Figure 2c). Notably, the infant T-cell receptor repertoire is restricted,65 and only a minor fraction of infant T cells + + IgM-only and IgM IgD memory B cells are capable of mounting typical Th2 responses.66 Similarly, a lower Two distinct subpopulations of human IgM-expressing PB B cells replication history of IgM+IgD+CD27+ versus GC B cells44 is with somatically mutated IgV genes exist, IgM-only B cells expected for memory B cells that leave the GC early. Moreover, (IgM+IgDlow/ − ) and IgM+IgD+ B cells, both expressing CD27 and it is unclear whether hyper-IgM II patients may retain in particular representing ∼ 5% and 15% of B cells, respectively, in PB and tissues residual GC activity that gives rise to lowly mutated secondary lymphoid organs, hence representing a major fraction IgM+IgD+CD27+ B cells. Indeed, other ligands than CD40L can of the B-cell pool.2,17,49 trigger CD40 signaling.67,68 Finally, regarding the presence of Whereas a GC derivation of IgM-only B cells is accepted,30 the mutated IgM+IgD+CD27+ B cells in the fetus, there is hardly any origin of IgM+IgD+CD27+ B cells is disputed.50 It was proposed that information of whether (primitive) GC reactions may occur even if the latter represent naive B cells that acquired IgV gene mutations no infection is detectable. as a means of primary BCR diversification,30 as it occurs among A major weakness of the hypotheses of a TI generation of animal species with restricted IgV recombinatorial diversity.51 IgM+IgD+CD27+ B cells is that no candidate histological structure The strongest argument for such a scenario was that hyper-IgM has been identified in any human where this would occur. As SHM patients with a nonfunctional CD40L gene, lacking typical GC is strictly dependent on AID expression,7 such structures would structures, possess IgV gene mutated IgM+IgD+CD27+ B cells.52 have to contain AID-expressing B cells that are indeed detectable The presence of some lowly mutated IgM+IgD+CD27+ B cells in in secondary lymphoid organs and the outside of fetal liver and cord blood also supported this idea.52,53 Moreover, GC.69,70 However, in lymph nodes and mucosa-associated molecular analyses indicated that IgM+IgD+CD27+ B cells underlie lymphatic tissues, these are singular cells that likely undergo generation or selection mechanisms distinct from IgG memory B AID-dependent class switching. In the bone marrow, a recent cells, indicated by reduced IgV ,54 distinct IgV mouse study indicates that rare AID+ B cell precursors are destined gene repertoire,29 an early diversified BCR repertoire in infants55 as to undergo apoptosis.69 To generate an IgM+IgD+CD27+ B-cell well as a reduced proliferation history.44 population with 2–4% IgV mutations, multiple rounds of A second scenario proposed the generation of IgM+IgD+CD27+ proliferation and mutation are needed, and a histological B cells in TI immune responses. This was based on their reported microenvironment allowing selection is essential as most muta- absence in an asplenic human and their vanishing after tions are disadvantageous or may cause autoreactivity. Indeed, in splenectomy,56 because the , and in particular splenic sheep and rabbits, SHM during primary BCR diversification occurs marginal zone B cells, are considered to play an important role in in specialized histological structures composed of large B-cell TI immune responses. However, other studies did not confirm this follicles.51 However, in humans no follicular AID+ non-GC specific dependency and showed that IgG memory B cells are also structures have been identified, although numerous immunohis- reduced after splenectomy.57–59 tological studies have been performed.70–73 In contrast to the proposed GC-independent generation, A final consideration, however, may resolve much of the numerous observations support the idea that IgM+IgD+CD27+ B controversial discussion about the origin of IgM+IgD+CD27+ B cells are post-GC memory B cells. First, IgM+CD27+ and IgG+ B cells cells. The strongest indications for a GC-independent generation share typical memory B-cell functions,22,25,33,60 a common PC of IgM+IgD+CD27+ B cells derive from studies with fetal or infant differentiation propensity,21,33,36 and they frequently reenter GC tissues. In all of these instances, most of the IgM+IgD+CD27+ B reactions upon secondary challenge (Figure 2a),27,33 as was also cells detected were unmutated, and the mutated ones had shown for murine IgM memory B cells.31 Second, they accumulate typically only few mutations.52,53,55 Perhaps, there is a low level of with age because of cumulative immune responses.36,52 Third, SHM activity that can occur during ontogenetically early B-cell they are similarly long-lived as class-switched memory B cells.61 development or during marginal zone B-cell generation, without Fourth, they have a transcriptome profile identical to IgM-only the need for extensive proliferation, mutation and selection.

© 2016 Macmillan Publishers Limited, part of Springer Nature. Leukemia (2016) 2283 – 2292 Human memory B cells M Seifert and R Küppers 2288 During childhood and adolescence, however, the IgM+IgD+CD27+ Many of these bona fide memory B-cell subsets are associated with B-cell pool becomes dominated by post-GC memory B cells. specific disease conditions, as reviewed elsewhere in more detail.90 Memory B cells displaying low to negative CD21 Splenic marginal zone B cells expression and lack of CD27 have been described in human + tissues near epithelial surfaces and PB. The tissue-resident The spleen contains a highly specialized population of IgM B cells − CD21lowCD27 B cells were described as FCRL4+, mostly class located in the marginal zones surrounding endothelial venules switched and IgV gene mutated.91 They secrete antibodies and and primarily involved in immune responses against blood-borne proliferate upon stimulation by T cell-derived , but not pathogens. Whereas murine splenic marginal zone (sMGZ) B cells 91 low − BCR stimulation. In contrast, CD21 CD27 B cells in the PB represent a separate, largely Ig-unmutated and GC-independent were mainly associated with age, autoimmune conditions or B-cell lineage,74 human sMGZ B cells are different in that they are 57,75–77 particular infections and represent dysfunctional or exhausted mostly IgV gene mutated, express CD27, show mutations in 92,93 77,78 memory B cells. These cells were originally also characterized the BCL6 gene and enhanced immune response potential. This + + + + as FCRL4 B cells, but recent studies indicate that they are more high similarity to IgM IgD CD27 memory B cells has led to a likely FCRL5+.94,95 similar debate of whether these are post-GC B cells or derive from + + + − 30,57 Human CD20 CD27 CD43 CD70 B cells have been proposed independent generation pathways. Human sMGZ seem to to represent the human counterpart of murine B1 B cells.96 play a key role in TI immune responses, for example, against fi 30,56 However, this nding is debated and more detailed analyses encapsulated bacteria. sMGZ B cells may be composed of at revealed that this minor B-cell population rather represents least two potentially distinct subsets, one exhibiting mutated IgV 97 + − 55,79,80 plasmablasts. Finally, a small fraction of IgM CD27 memory B genes and the other lacking such signs of GC experience. cells was described showing very low levels of IgV gene mutations Moreover, in the spleen of young children, a specific precursor 98 81 and lacking expression of the naive B-cell marker ABCB1. subset of sMGZ B cells has been described. To merge these Supporting this notion, our own studies revealed a subset of seemingly contradictory findings, it was proposed that more than + low − − 75,79–81 IgM IgD CD27 CD23 B cells with mostly unmutated IgV and one subset of human sMGZ B cell exists. BCL6 genes (own unpublished observations).99 Human sMGZ B cells can interact with that reside nearby the MGZ, show B-cell helper function and induce class switching,82 although another study contradicts these findings.83 THE COMPLEXITY OF THE HUMAN MEMORY B-CELL A similar functional connection between PB neutrophils and the REPERTOIRE + + + proposed circulating sMGZ counterpart, that is, IgM IgD CD27 B Next-generation sequencing of BCR repertoires has become a cells, has been shown by in vitro studies.33 + powerful way to determine the molecular diversity of B-cell In addition to the sMGZ, several further IgM B cell-rich areas subsets. Initial next-generation sequencing analyses provided first have been described, including the subepithelial area of the , large-scale insights into mutation load, IgV gene usage, junctional intestinal Peyer’s patch dome regions, the subcapsular sinus of 29,50,100–102 84 diversity and clonal expansions among human B cells. lymph nodes and the thymic medulla. However, it is unclear However, these studies were restricted for a comprehensive whether these subsets represent distinct memory B-cell entities or characterization of the human memory B-cell pool, because too rather typical IgM memory B cells that populated MGZ equiva- few B cells or memory subsets were analyzed. In addition, these lents, suggesting that similar recruitment and retention mechan- studies collected IgV sequences as single reads, that is, without isms for post-GC memory B cells also occur in these tissues. ‘coverage’ or replicate analysis. Therefore, a reliable quantification Taken together, in young children, there is indication for a sMGZ of clonal expansions is hampered, as amplificates from the same B-cell population with a distinct developmental pathway, but in cell cannot be distinguished from truly expanded clone members, + adults, it appears that the sMGZ is dominated by mostly IgM and PCR errors may be misinterpreted as intraclonal diversity. post-GC B cells. A first analysis of human memory B-cell subsets described a surprisingly different Ig heavy chain V gene repertoire between IgD-only memory B cells IgM+IgD+CD27+ and class-switched memory B cells, postulating that these populations develop in response to different A peculiar and very rare subset of memory B cells are IgD-only B 29 + − + + cells, accounting for 0.1–0.5% of PB B cells in ∼ 10% of individuals. antigens. A subsequent analysis of IgG CD27 and IgG CD27 memory B cells revealed a frequent clonal relationship of these IgD-only B cells are unique in that they have undergone an 39 μ subsets, but also different CDRIII properties. unusual class-switch recombination, leading to deletion of the C + + δ 85 In our own next-generation sequencing study of IgM IgD gene but retention of C . Hence, they can only express IgD. + + + + − These cells show the highest IgV gene mutation load of all human CD27 , IgM-only, IgG CD27 and IgG CD27 memory B cells, ∼ λ covering 40 000 and 66 000 individual B cells from two donors B cell subsets (average frequency of 12%), 90% express light ⩾ chains (versus 40% for all other B cells) and they have a highly ( 2-fold coverage, replicate analysis) revealed that many memory biased IGHV gene segment usage.85,86 All of these features fitto B cells could be assigned to expanded, often very large clones (sometimes 4100 000 members estimated in PB) with often the hypothesis that IgD-only B cells are generated in 27 86 enormous intraclonal mutation diversity. Importantly, both IgM- -driven GC reactions. In line with this idea, IgD- + + + ∼ 87 only and IgM IgD CD27 B cells frequently belonged to clones only GC are found in 10% of , and some bacteria + causing upper respiratory tract infection, such as Moraxella also encompassing IgG memory B cells (Figure 2b). The larger a clone, the higher was the chance to find both switched and catarrhalis, express an IgD-binding that can serve as 27 88 nonswitched members. This analysis suggested that practically superantigen. Although IgD-only B cells in the PB are very rare, all GC reactions producing IgG+ B cells also give rise to IgM+(IgD+) ∼ 15% of tonsillar PCs represent IgD-only cells, and the low memory B cells, hence largely reflecting the murine situation.31,103 amount of IgD detectable in human serum is presumably derived This confirmed and extended an earlier analysis, where we from IgD-only PCs, as secreted IgD mostly contains λ light revealed a frequent clonal relationship and hence common GC chains.89 origin of IgM+IgD+CD27+ and IgG+ B cells by clone-specific PCR.28 Many large memory B-cell clones were not only complex in Other memory B-cell subsets terms of composition by IgM+(IgD+) and IgG+ memory B cells and According to surface molecule expression or tissue localization, intraclonal IgV gene diversity, but also by IgG clone members further human memory B-cell subsets have been described. expressing various IgG subclasses and involving CD27+ as well as

Leukemia (2016) 2283 – 2292 © 2016 Macmillan Publishers Limited, part of Springer Nature. Human memory B cells M Seifert and R Küppers 2289 CD27− IgG memory B cells. This shows that from one GC B-cell and paralleled by their gradual acquisition of IgV gene clone, various types of memory B cells, and thus very large, mutations.99 complex and diverse memory B-cell clones can be generated. (Figure 2b). A key finding of our human memory BCR repertoire HUMAN MEMORY B CELLS AND LYMPHOMAS analyses was the dynamical aspect of on average early IgM Most human B-cell malignancies are derived from GC B cells. This versus late IgG memory B-cell generation during a GC reaction is likely linked to the vigorous proliferation of GC B cells, their (Figures 1 and 3).27,28 Hence, the generation of IgM+IgD+CD27+ reduced surveillance for genomic integrity and the Ig gene memory B cells occurs on average before that of IgM-only B cells remodeling processes in the GC (SHM and class switching) that (that probably downregulated IgD because of extended activa- can cause chromosomal translocations and as a side effect of SHM tion) and both IgM memory B-cell subsets on average before class- also mutations in non-Ig genes.5,104 However, a number of B-cell switched CD27+ memory B cells. This finding is also reflected by and lymphomas show phenotypic and genetic features the dendrogram structures that are more diverse for IgM than IgG that resemble memory B cells, and therefore a derivation of these members (Figures 2a and c). Thus, the character of a newly tumors from memory B cells is proposed. These malignancies generated memory B cell is partly determined by the duration of include a subset of chronic lymphocytic leukemia (CLL) with its GC participation. Of note, this time-linked generation is likely to mutated IgV genes,105,106 nodal MGZ lymphomas,107,108 hairy cell occur not in separate phases but rather in a convergent manner, leukemia,109 some mantle cell lymphomas110 and some sMGZ as indicated by rare IgM memory B cells among highly mutated lymphomas.111 A hallmark of these lymphomas is the presence of (late) cells and some class-switched memory B cells among lowly somatically mutated IgV genes (and also BCL6 mutations, where mutated (early) post-GC B cells (the latter with a tendency to this has been analyzed), suggesting a GC experience, but mostly a express CD27 less frequently) (Figures 2 and 3). lack of expression of GC B-cell markers or ongoing SHM. Note that The concept that memory B-cell subsets are closely related, but some CLL cases may show signs of a low level of ongoing IgV mostly generated in consecutive phases of GC reactions, is gene diversification.112 The close resemblance of cells supported by a recent epigenome analysis.99 That study revealed in mutated CLL and hairy cell leukemia to memory B cells is mainly a clear, linear arrangement of memory B-cell subsets according to based on phenotypic and transcriptome similarities,105,106,109 and an epigenetical reprogramming pattern that is stepwise acquired also IgV gene mutation analysis.113 For CLL, sMGZ and other MGZ and intensified in the course of a GC reaction. The sequence of lymphomas, there is indication for autoreactivity or in some human memory B-cell subsets along this reprogramming axis is instances specificity for infectious agents of the BCR expressed by identical to their average position in genealogical dendrograms the lymphoma cells.113,114 Thus, such lymphoma cells might be

Figure 3. Establishment of complex and diverse memory B-cell clones during the GC reaction. GC B cells undergo multiple rounds of proliferation, mutation and selection. This leads to a stepwise increase in the IgV gene mutation load and also class switching. Memory B cells are generated throughout the GC reaction. Arrows indicate acquisition of IgV gene mutations, and red crosses indicate apoptosis. Cell clusters indicate proliferation without additional mutations. Based on the mutation patterns, genealogical trees reconstruct the continuous generation of memory B cells during the GC response, demonstrate the large memory B-cell diversity and show that IgM+(IgD+) and class-switched memory B cells often derive from the same GC B-cell clone.

© 2016 Macmillan Publishers Limited, part of Springer Nature. Leukemia (2016) 2283 – 2292 Human memory B cells M Seifert and R Küppers 2290 continuously triggered for proliferation, contributing to lymphoma 8 Pasqualucci L, Migliazza A, Fracchiolla N, William C, Neri A, Baldini L et al. clone survival and expansion. BCL-6 mutations in normal germinal center B cells: evidence of somatic hyper- Although the B-cell lymphomas discussed here resemble mutation acting outside Ig loci. Proc Natl Acad Sci USA 1998; 95: 11816–11821. memory B cells, it may well be that most of the transforming 9 Victora GD, Nussenzweig MC. Germinal centers. Annu Rev Immunol 2012; 30: – events involved in their pathogenesis were acquired at earlier 429 457. 10 Shapiro-Shelef M, Calame K. Regulation of plasma-cell development. Nat Rev stages of their development. In CLL, some initial genetic lesions 5 – 115 Immunol 2005; :230 242. are even acquired at the stage of hematopoietic stem cells, and 11 Gitlin AD, von Boehmer L, Gazumyan A, Shulman Z, Oliveira TY, Nussenzweig MCL and MGZ lymphomas frequently carry chromosomal MC. Independent roles of switching and hypermutation in the development and translocations that likely happened as mistakes during early B-cell persistence of B memory. Immunity 2016; 44: 769–781. 104 development or in the GC. 12 Basso K, Klein U, Niu H, Stolovitzky GA, Tu Y, Califano A et al. Tracking CD40 Notably, most cases of the B-cell lymphomas and leukemias signaling during germinal center development. Blood 2004; 104: 4088–4096. with a presumed memory B-cell origin express IgM ( ± IgD), and 13 Maarof G, Bouchet-Delbos L, Gary-Gouy H, Durand-Gasselin I, Krzysiek R, have not undergone class switching (hairy cell leukemia is an Dalloul A. Interleukin-24 inhibits the plasma cell differentiation program in 115 – exception to this).116 The reason for this is largely unclear. human germinal center B cells. Blood 2010; :1718 1726. 14 Shinnakasu R, Inoue T, Kometani K, Moriyama S, Adachi Y, Nakayama M et al. However, if one considers that IgM memory B cells have a higher Regulated selection of germinal-center cells into the memory B cell compart- propensity to differentiate into proliferating cells upon activation, ment. Nat Immunol 2016; 17:861–869. whereas class-switched memory B cells tend to differentiate 15 Kaji T, Ishige A, Hikida M, Taka J, Hijikata A, Kubo M et al. Distinct cellular preferentially into resting PC (discussed above), IgM memory B pathways select germline-encoded and somatically mutated antibodies into cells might be at an increased risk to transform into continuously immunological memory. J Exp Med 2012; 209:2079–2097. proliferating lymphomas. 16 Taylor JJ, Pape KA, Jenkins MK. A germinal center-independent pathway generates unswitched memory B cells early in the primary response. J Exp Med 2012; 209: 597–606. CONCLUDING REMARKS 17 Klein U, Küppers R, Rajewsky K. Evidence for a large compartment of IgM- 89 – Integrating the molecular evidences from Ig heavy chain expressing memory B cells in humans. Blood 1997; : 1288 1298. 18 Macallan DC, Wallace DL, Zhang Y, Ghattas H, Asquith B, de Lara C et al. repertoire analysis, clonal relation, Ig and BCL6 mutation patterns, B-cell kinetics in humans: rapid turnover of peripheral blood memory cells. Blood genealogical dendrogram analysis and epigenome profiling, we 2005; 105: 3633–3640. propose a model for memory B-cell generation where these cells 19 Crotty S, Felgner P, Davies H, Glidewell J, Villarreal L, Ahmed R. Cutting edge: are generated exclusively in and continuously throughout GC long-term B cell memory in humans after smallpox vaccination. J Immunol 2003; reactions. From single GC B-cell clones various types of memory B 171: 4969–4973. cells with specific functions and highly diversified clone members 20 Yu X, Tsibane T, McGraw PA, House FS, Keefer CJ, Hicar MD et al. Neutralizing are generated. Notably, the duration of the GC participation seems antibodies derived from the B cells of 1918 influenza pandemic survivors. Nature 455 – to be a major determinant for these distinct memory B-cell 2008; : 532 536. fi 21 Bernasconi NL, Traggiai E, Lanzavecchia A. Maintenance of serological memory phenotypes (Figure 1). This way, high-af nity and diverse memory 298 fi by polyclonal activation of human memory B cells. Science 2002; : B cells are generated and at the same time further diversi cation 2199–2202. potential of the memory B-cell pool is preserved. Thus, 22 Good KL, Avery DT, Tangye SG. Resting human memory B cells are intrinsically this memory B-cell generation system provides an additional programmed for enhanced survival and responsiveness to diverse stimuli level of flexibility and selection potential to the adaptive compared to naive B cells. J Immunol 2009; 182:890–901. immune system. 23 Avery DT, Deenick EK, Ma CS, Suryani S, Simpson N, Chew GY et al. B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long- lived antibody responses in humans. J Exp Med 2010; 207: 155–171. CONFLICT OF INTEREST 24 Bernasconi NL, Onai N, Lanzavecchia A. A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and con- The authors declare no conflict of interest. stitutive expression in memory B cells. Blood 2003; 101: 4500–4504. 25 Good KL, Tangye SG. Decreased expression of Kruppel-like factors in memory B ACKNOWLEDGEMENTS cells induces the rapid response typical of secondary antibody responses. Proc Natl Acad Sci USA 2007; 104: 13420–13425. We thank Bettina Budeus for support. Own work discussed in this review was 26 Engels N, König LM, Heemann C, Lutz J, Tsubata T, Griep S et al. Recruitment of supported by the Deutsche Forschungsgemeinschaft through Grants Ku1315/8-1, the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor- GRK1431/2 and SE1885/2-1. We apologize to those colleagues whose primary work intrinsic costimulation to class-switched B cells. Nat Immunol 2009; 10: could not be cited because of reference restrictions. 1018–1025. 27 Budeus B, Schweigle de Reynoso S, Przekopowitz M, Hoffmann D, Seifert M, Küppers R. Complexity of the human memory B-cell compartment is determined REFERENCES by the versatility of clonal diversification in germinal centers. 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