Memory B cells. Tomohiro Kurosaki 1,2, Kohei Kometani 2,3 and Wataru Ise 1

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1 Memory s Tomohiro Kurosaki 1,2, Kohei Kometani 2,3 and Wataru Ise 1 Abstract The immune system can remember a previously experienced pathogen and can evoke an enhanced response to reinfection that depends on memory lymphocyte populations. Recent advances in tracking antigen-experienced memory s have revealed the existence of distinct classes of cells that have considerable functional differences. Some of these differences seem to be determined by the stimulation history during memory cell formation. To induce rapid recall antibody responses, the contributions of other types of cells, such as memory T follicular helper cells, have also now begun to be appreciated. In this Review, we discuss these and other recent advances in our understanding of memory s, focusing on the underlying mechanisms that are required for rapid and effective recall antibody responses. 1 Laboratory of Lymphocyte Differentiation, World Premier International Immunology Frontier Research Center and Graduate School of Frontier Biosciences, Osaka University, 3 1 Yamada-oka, Suita, Osaka , Japan. 2 Laboratory for Lymphocyte Differentiation, RIKEN Center for Integrative Medical Sciences, Tsurumi ku, Yokohama, Kanagawa , Japan. 3 Present address: Department of Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg 79108, Germany. Correspondence to T.K. e mail: kurosaki@ifrec.osaka u.ac.jp doi: /nri3802 Published online 13 February 2015 Secreted neutralizing antibodies are of central importance to immune protection of the host, particularly against acutely cytopathic viruses such as vesicular stomatitis virus or influenza virus. Therefore, the success of humoral memory depends on at least two layers of defence. Pre-existing protective antibodies secreted by long-lived plasma cells function as a first line of defence against reinfection if the antibody concentration at the site of reinfection is sufficiently high (constitutive humoral memory). If constitutive memory is not sufficient, a second line of defence becomes functional; pathogen-experienced memory s are rapidly reactivated to produce antibodies (reactive humoral memory). Compared with the primary antibody response, the reactive humoral memory response is typically faster, of greater magnitude and consists of antibodies of switched isotypes and higher affinity 1. The improved responsiveness and the maintenance of such heightened responsiveness have been thought to be primarily due to the activity of small numbers of memory s that were generated during the primary immune response to the antigen and that subsequently survived. Thus, characterizing the properties of these memory s and determining how these unique properties are generated are of fundamental interest to understanding the basis of humoral memory. As the antibodies that are newly generated following secondary antigen challenge are of switched isotypes and have higher affinity than the antibodies generated in the primary response, it has been thought that the functionally important memory s derive their efficacy from class-switched and high-affinity receptors (BCRs) on their surface (for example, IgG + memory s), which develop within the germinal centre 2 4. However, recent evidence has shown the existence of germinal centre-independent memory s 5 7 and of unswitched IgM + memory s 8 10, in addition to the germinal centre-dependent switched memory s. Therefore, questions arise about whether these newly identified memory subsets have an important, possibly distinct, role in humoral immunity that depends on features such as longevity, rapid responsiveness and affinity and, if so, how these distinct roles are engendered. In this Review, we summarize and discuss recent progress with regard to these and related questions, with the aim to update the classical view of memory biology. We specifically emphasize mouse studies, as new aspects of memory s have been uncovered following the development of novel mouse models in the past several years. It is not yet clear whether all of the same principles hold true for memory s in humans. We first discuss the pathways that generate memory s and the heterogeneity of these cells, and then we focus on the mechanisms that underlie rapid and efficient memory responses. Generation of T cell-dependent memory s In T cell-dependent responses (FIG. 1), accumulating evidence shows that antigen-activated proliferating s (FIG. 1a) begin to follow one of three fates by differentiating into extrafollicular short-lived plasma cells (FIG. 1b), germinal centre-independent memory s (FIG. 1c) or germinal centre-dependent memory s 11 (FIG. 1e). In this pre-germinal centre period, isotype switching (for example, to IgG) but not somatic hypermutation begins to occur. NATURE REVIEWS IMMUNOLOGY VOLUME 15 MARCH

2 REVIEWS Secondary lymphoid organ follicle FDC Affinity selection T FH cell e Germinal centre-dependent humoral memory T cell zone Antigen BCR Germinal centre d Light zone Dark zone Clonal expansion and BCR diversification Memory f Serological memory Naive Antigenmediated activation c Germinal centreindependent humoral memory Long-lived plasma cell Memory T FH cell Dendritic cell MHC class II Antigen TCR Naive CD4 + T cell Antigenmediated activation a proliferation b Antibody Extrafollicular foci of antibody production Short-lived plasma cell Isotype switching A switch recombination in DNA that encodes the constant region of the immunoglobulin heavy chain, from IgM to any of IgG, IgA or IgE. The recombination occurs in repetitive DNA sequences (switch regions) that are located upstream of each constant region gene. Figure 1 T cell-dependent memory generation. Antigen-activated s and T cells migrate towards the borders of the follicles and the T cell zones of secondary lymphoid organs, respectively, which leads to them establishing stable T cell interactions and enables s to receive helper signals from cognate CD4 + T cells. Activated s and T cells then migrate to the outer follicles, where s undergo proliferation (part a). Some of the proliferating s differentiate into short-lived plasma cells (part b), which give rise to the extrafollicular foci, and some develop into memory s (part c; germinal centre-independent memory s). Alternatively, the activated s can return to the follicle and can undergo rapid proliferation to form the germinal centre (part d). In the dark zone of the germinal centre, the clonal expansion of antigen-specific s is accompanied by receptor (BCR) diversification through somatic hypermutation. The s that exit the cell cycle relocate to the light zone, where affinity selection takes place through interaction with immune complex-coated follicular dendritic cells (FDCs) and antigen-specific T follicular helper cells (T FH cells). The affinity-matured germinal centre s can re enter the germinal centre cycle. Alternatively, these germinal centre s exit the germinal centre, either as memory s (part e; germinal centre-dependent memory s) or as long-lived plasma cells (part f) that contribute to serological memory. The strength of signals that s receive is likely to determine their fate; stronger signals (indicated by bold arrows) favour development into plasma cells or germinal centre s, whereas weaker signals (indicated by narrow arrows) determine memory differentiation. TCR, T cell receptor. Somatic hypermutation A process in which point mutations are generated in the variable regions of immunoglobulin genes, thus creating a more specific repertoire when combined with selection. Some mutations might increase the affinity of the receptor (BCR) for the specific antigen, but others might lead to a loss of antigen recognition by the BCR or to the generation of a self-reactive receptor. Germinal centre-independent memory s. Insights into the mechanisms that affect the fate decision of proliferating activated s between becoming germinal centre-independent memory s (FIG. 1c) and entering germinal centre differentiation (FIG. 1d) have been provided by recent dynamic imaging studies. By using competition experiments between s with high and low affinity for the hapten 4 hydroxy-3 nitrophenylacetyl (NP), it was shown that high-affinity s presented more peptide MHC to cognate T follicular helper cells (T FH cells) at the T cell border in the lymph node or in the spleen; high-affinity s thereby made longer-lasting contact with the T FH cells, obtained more T cell help and were more likely to differentiate into germinal centre s 12. Therefore, these data indicate that affinitydependent selection occurs at the T cell border as a result of T cell help, which could affect fate decisions 13. The requirement for durable T FH cell conjugate formation before a can enter the germinal centre reaction has also been shown in mice that are deficient for signalling lymphocyte 150 MARCH 2015 VOLUME 15

3 Box 1 Metabolic reprogramming during the transition from effector to memory lymphocytes Initial studies of the metabolic reprogramming of lymphocytes were carried out using CD8 + T cells. In contrast to effector CD8 + T cells, which mainly use glycolysis to form ATP and NADH, and which have a reduced mitochondrial mass (indicative of anabolic metabolism), memory CD8 + T cells maintain a greater mitochondrial mass, which supports mitochondrial fatty acid oxidation (catabolic metabolism) 82. This enhanced mitochondrial function probably provides memory T cells with a bioenergetic advantage and rapidly fuels their subsequent reactivation upon re-encountering antigen. In addition, during the transition from the effector to the memory phase of the response, limited levels of oxygen, nutrients, growth factors and other signals occur in a metabolically restrictive environment. This leads to decreased levels of ATP, thereby triggering the activation of AMP-activated protein kinase (AMPK), which suppresses the anabolic pathway and promotes the catabolic pathway. Indeed, activation of AMPK by the administration of metformin enhances the generation of CD8 + memory T cells 83. AMPK is known to be a potent activator of autophagy. Considering the importance of autophagy for the generation and survival of memory s 84, metabolic reprogramming probably also occurs during the contraction phase of the effector response in the development of memory s 84. This might provide an explanation for the previously observed phenotypes of mice that are deficient for the cytokine interleukin-21 or its receptor; both types of mice had an accumulation of IgG1 + memory s, as well as a reduction in germinal centre cell numbers 85. Germinal centre s use continuous glycolysis for their maintenance, thereby requiring high levels of cytokines as growth factors. Assuming that metabolic reprogramming from anabolic to catabolic metabolism takes place for s as well as for T cells, memory s may not require high levels of cytokines for their maintenance. T follicular helper cells (T FH cells). A distinct subset of antigen-activated CD4 + T cells expressing CXC-chemokine receptor 5 and lymphoma 6. T FH cells are essential for germinal centre formation and regulate the activation and function of germinal centre s. B2 cells The major and conventional population in humans and mice. Marginal zone and follicular s belong to the B2 cell lineage and arise from bone marrow precursor cells. B1 cells A self-renewing subset of mature s that predominates in the peritoneal and pleural cavities. B1 cells recognize self components, as well as common bacterial antigens, and are primarily responsible for the production of natural serum IgM. activation molecule-associated protein (SAP); deficiency of SAP shortens the duration of T cell conjugates in the first 2 days after antigen exposure and markedly decreases the number of germinal centre s 14. So, how are early germinal centre-independent memory s generated (FIG. 1c)? In vivo transfer experiments have shown that memory s differentiate from an activated precursor cell expressing CD38 and GL7 (REFS 6,7). Among the various signals provided by T cells, the CD40 signal alone can induce activated s to differentiate down the memory pathway but not into germinal centre cells 6. In addition to the CD40 signal, cytokine signalling is probably required for germinal centre differ entiation. Indeed, interleukin 21 (IL 21) was shown to upregulate the expression of lymphoma 6 (BCL 6) in s, which is a crucial transcription factor for germinal centre formation and maintenance 15,16. Taking these data and the imaging data into account, the following model might be suggested: the formation of durable T FH cell conjugates to provide adequate T cell help enables s to differentiate into germinal centre s (FIG. 1d). However, if the duration of conjugate formation is fairly short, s are more likely to join the germinal centre-independent memory pool (FIG. 1c). Given that class switching but not somatic hypermutation occurs during this early period, BCR specificities of the germinal centre-independent memory s are likely to reflect those of the initial responding s, although some selection could be imposed by cognate T cells during priming to germinal centre-independent stages. Therefore, the germinal centre-independent memory s may enable the host to maintain a broad range of antigen-specific s, at least compared with the germinal centre-dependent memory s described below. This could provide protection against pathogens that bear related but distinct antigens and epitopes. Germinal centre-dependent memory s. Recent excellent studies have revealed the dynamics of the germinal centre reaction and these are reviewed elsewhere 22 in this Focus issue. Therefore, in this Review, we emphasize the mechanisms by which germinal centre s are selected as memory s. Although the precise mechanism is still unclear, one hypothesis is that there is a master regulator of transcription that directs the cells towards a memory fate. However, despite extensive analysis of gene expression profiles, no single deterministic transcription factor for memory s has been elucidated. Thus, a more prevailing alternative idea is that memory s differentiate stochastically from germinal centre s and that a survival advantage is sufficient for memory differentiation. In support of this hypothesis, forced expression of the pro-survival factor BCL 2, or deletion of the pro-apoptotic factors BCL-2-interacting mediator of cell death (BIM; also known as BCL-2L11) or p53-upregulated modulator of apoptosis (PUMA; also known as BBC3), leads to an increase in the size of the IgG1 + memory compartment 23,24. Consistent with these observations, the expression levels of BIM and BCL 2 are decreased and increased, respectively, in IgG1 + memory s compared with activated s 7,25. In relation to the survival advantage of memory s, the importance of metabolic reprogramming at the transition between actively proliferating (effector) cells and quiescent (memory) cells is becoming more widely appreciated (BOX 1). Generation of T cell-independent memory s It was previously assumed that memory s are only formed during T cell-dependent immune responses usually in response to protein antigens and therefore that conventional B2 cells are the exclusive participants in memory generation. However, recent data clearly show that B1 cells can also generate memory s during T cell-independent immune responses B1 cells are the most abundant s in the peritoneal cavity but they are also present at a low but detectable frequency in the spleen 29. B1 cells are subdivided into the B1a (CD5 + ) and B1b (CD5 ) subsets. In the case of B1a cells, priming with a glycolipid (FtL) derived from Francisella tularensis live vaccine strain elicits long-term FtL-specific memory B1a cells (mainly IgM + ) that NATURE REVIEWS IMMUNOLOGY VOLUME 15 MARCH

4 REVIEWS persist in the peritoneal cavity, but not elsewhere, in a T cell-independent manner 28. Upon FtL rechallenge, co stimulation with a Toll-like receptor 4 (TLR4) agonist is required for plasma cell differentiation 28. This rechallenge seems to induce the migration of the peritoneal cavity-localized memory B1a cells to the spleen, where differentiation into plasma cells takes place. These data indicate that the microenvironments necessary for the maintenance and the activation of memory B1a cells could differ. In the case of B1b cells, by tracing antibody responses against Streptococcus pneumoniae and Borrelia hermsii, these cells were shown to generate memory s and to persist mainly in the peritoneal cavity, similarly to memory B1a cells 26,30. In a more recent study 27, s specific for NP Ficoll (which mimics a carbohydrate antigen) were traced and analysed; the memory B1b cells that were generated retained many of the phenotypic characteristics of naive B1b cells in terms of longevity and sensitivity to antigenic stimulation. Therefore, the fundamental features of antigen-specific memory B1 cells seem to resemble those of the naive s from which they derive. In summary, there are multiple pathways for generating memory. Although T cell-independent memory s can be generated as discussed above, it seems that their recall response is quantitative, rather than qualitative, in nature. Thus, aside from the increased frequency of antigen-specific s, it is unclear whether T cell-independent memory s have an intrinsic advantage compared with their naive counterparts to respond more rapidly and more robustly to antigen, as is seen in T cell-dependent memory. Therefore, we hereafter focus on several aspects of canonical, T cell dependent memory s. Heterogeneity of memory s As mentioned above, during the primary immune response, several types of memory s are generated in a spatiotemporal manner, which evokes the idea that these memory s have distinct functions 31. The development of perpetual labelling techniques for all types of antigen-experienced s 9,32 is now enabling the identification and the functional characterization of various types of memory s, including IgM + memory s. IgM + memory s. Two decades ago, it was hypothesized that there are two distinct types of memory s IgM + and IgG + cells which are activated and function in a distinct manner during reinfection 33. However, this idea was not experimentally tested mainly because of the absence of appropriate markers and methods to distinguish IgM + naive s from IgM + memory s in the mouse system. Two groups have recently addressed this question and, in support of the original hypothesis, they have reached a similar conclusion that upon antigen rechallenge, IgG + memory s preferentially differentiate into plasmablasts, whereas IgM + memory s proliferate more and enter the germinal centre reaction 9,10. However, it seems that there is functional heterogeneity even within the IgM + or IgG + memory pools, and it cannot be excluded that IgG + memory s can re enter germinal centres or that IgM + memory s might produce a plasmablast response. A more recent study has proposed that other markers (CD80 and programmed cell death 1 ligand 2 (PDL2)) are more functionally relevant to memory subsets; CD80 PDL2 memory s enter the germinal centre reaction, whereas CD80 + PDL2 + memory s promptly differentiate into plasmablasts upon restimulation 34. IgG + and IgA + memory s. The above-mentioned studies mainly used memory s expressing IgG1 (in the case of NP chicken-γ globulin plus alum as the antigen) or expressing mixtures of IgG1, IgG2a and IgG2b (in the case of sheep red blood cells or phycoerythrin plus complete Freund s adjuvant as antigens). However, a recent study shows the need to functionally characterize each isotype of memory 35. Transcription factors that are induced in s by cytokines are important for regulating subsequent behaviour in the primary response; for example, interferon γ (IFNγ)-induced T bet (also known as TBX21) expression is known to be important for IgG2a class switching. Interestingly, such transcription factors are also important for the survival of immunoglobulin class-specific memory s 35. Expression of T bet or retinoic acid receptor-related orphan receptor-α (RORα) in IgG2a + or IgA + memory s, respectively, is higher than in naive s, and these transcription factors are crucial for memory cell survival, probably by controlling the transcription of genes that encode cell-surface BCR components 35. As each subclass of immunoglobulin has unique biological activities as a result of its Fc portion, targeting particular transcription factors for developing antibody isotypeskewing vaccines could be an important strategy for immunotherapeutic applications. IgE + memory s. IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases 36. However, despite the biological importance of IgE memory, IgE + memory s have not been readily detected in vivo under physiological conditions. Using a monoclonal antigen-specific IgE + model, it was proposed that high-affinity IgE memory responses can be generated through sequential class switching of affinity-matured IgG1 + memory s 37. After rechallenge, IgG1 + memory s underwent class switching and differentiated into IgE-producing plasma cells. Two lines of evidence were provided that support this model: first, the presence of IgG1 switch region remnants in the immunoglobulin heavy chain locus of cells expressing high-affinity IgE 38 ; and second, defective production of high-affinity IgE antibodies in response to repeated immunization in IgG1 deficient mice 37. These data, together with recent data using sensitive IgE-reporter mice, strongly indicate that there is a lack of bona fide IgE-expressing memory s or that, if these cells do exist, they are only present in small numbers 39. By contrast, one study did identify IgE + memory s using IgE-reporter mice 40 ; however, caution is required in 152 MARCH 2015 VOLUME 15

5 interpreting these results, given that the reporter mice that were used contain additional sequences including an exogenous polyadenylation signal sequence that might alter normal IgE responses 40,41. In summary, these recent studies of memory s expressing IgM, IgG2a, IgA and IgE have shown that the origin, the function and the longevity of memory s could differ between cells expressing different antibody isotypes. Therefore, questions arise about how such heterogeneity is induced and whether different types of memory s are coordinately activated upon secondary infection. In particular, an apparent lack of bona fide IgE-expressing memory s requires us to revise the conventional concept that memory s expressing a particular class of immunoglobulin are responders for secondary memory responses of that class. Unique properties of memory s Key functional features of memory s are their longevity and their rapid and robust responses to antigen re exposure, which are the basis of vaccine success. In this section, we discuss recent progress in our understanding of the cellular and molecular bases for such properties of memory s. In particular, we focus on the mechanisms that underlie the maintenance, survival and robust responses of memory s, from a cell-intrinsic and a cell-extrinsic point of view. Stemness. Haematopoiesis is a well-known example of a biological system with long-term functions. In this system, the long-term maintenance of homeostasis depends on the co existence of somatic stem cells and more committed progenitor cells 42. The stem cells ensure the efficient replacement of more committed cells but at the same time maintain themselves through a process of selfrenewal. The more committed pro genitor cells can be quickly differentiated into more mature cells following exogenous stimulation. We postulate that such a stem cell-based mechanism might be similarly used by the humoral memory system, which requires bifunctionality to efficiently make effector cells upon re-encountering pathogens and simultaneously continue to maintain the responsive memory state. As IgG + memory s seem to have a greater propensity to differentiate towards plasma cells than IgM + memory s do, it could be suggested that the IgM + memory compartment contains more stem cell-like cells, whereas class-switched memory s, such as IgG + memory cells, correspond to committed progenitor cells (in other words, a more differentiated cell type). This proposal for memory s requires further study but would be akin to the situation for memory CD8 + T cells, for which substantial evidence of a stem cell-based model has recently been provided 43. Longevity. To determine which types of cells and which types of molecules are required for memory survival, previous studies have used IgG + memory s as a target. IgG + memory s can persist in the absence of T cells or input from precursor cells, but experiments using mice with follicular dendritic cells (FDCs) in which the gene encoding complement receptor 2 (Cr2) has been knocked out have suggested that there is a requirement for FDCs for the maintenance of IgG + memory s 44. In these mice, the primary IgG response was unaffected, but the secondary antibody response was significantly decreased. Notably, the impaired memory response corresponded with the reduced frequency of antigen-specific memory s. Thus, one straightforward interpretation is that CR2 on FDCs promotes the survival of IgG + memory s, directly or indirectly, possibly by functioning as a depot for antigen antibody complement complexes; however, the role of antigen persistence in memory responses is debated (see below). Inducible deletion of phospholipase Cγ2 (PLCγ2) after the generation of IgG1 + memory s substantially decreased the size of the memory compartment, which suggests a requirement for BCR signalling for IgG1 + memory survival 45. In terms of a requirement for antigen, genetic studies elegantly showed that cognate antigen was not necessarily required after the generation of IgG + memory s, which implicates a tonic-like BCR signal in the maintenance of IgG + memory s 46. Thus, the aforementioned requirement for T bet or RORα in IgG2a + or IgA + memory survival, respectively, can be accounted for by the fact that these transcription factors affect BCR expression 35. As a result, factors that participate in expression of the BCR components (class-specific immunoglobulin heavy and light chains, Igα and Igβ) and tonic BCR signalling molecules could be potential determinants of memory survival. BCR expression seems to control the expression of BCL 2 family proteins memory s upregulate cell survival factors such as BCL 2 and BCL 2A, and are sensitive to their broad inhibitors 47. The differential persistence of IgM + and IgG + memory s was recently shown; phycoerythrin-specific IgM + memory s persisted for 500 days after priming, whereas the number of IgG + memory s declined by many fold during this time period 10. This could be explained by differences in the self-renewal activity (stem cell-like nature) of IgM + and IgG + memory s (as discussed above) and/or by the existence of differential survival mechanisms. Consistent with the existence of differential survival mechanisms, blocking the receptors for -activating factor (BAFF; also known as TNFSF13B) and a proliferationinducing ligand (APRIL; also known as TNFSF13) did not affect the survival of IgG + memory s in vivo but had a marked effect on naive IgM + s 48. Preliminary results in mice suggest that IgM + memory s, similarly to naive s, require BAFF for their survival (T. Inoue and T.K., unpublished observations); therefore, the differential usage of BAFF and/or APRIL might be one cause of differential survival between IgM + and IgG + memory s in mice, although this requires further clarification and may not apply to human s. According to this hypothesis, a similar localization pattern of the IgM + memory s and naive s (scattered in the follicles) might be important 32. A recent NATURE REVIEWS IMMUNOLOGY VOLUME 15 MARCH

6 REVIEWS Fibroblastic reticular cells (FRCs). The most abundant population of nonhaematopoietic or stromal cells in T cell-rich areas of secondary lymphoid organs. FRCs facilitate interactions between T cells and dendritic cells, through the expression of cytokines and chemokines, such as interleukin-7, CC-chemokine ligand 19 (CCL19) and CCL21. study showed that a subset of fibroblastic reticular cells localized in the follicles produce BAFF, thereby establishing a favourable niche for controlling naive homeostasis 49. Although the use of phycoerythrin as a model antigen in mice clearly shows survival differences between IgM + and IgG + memory s, in humans, vaccinations and infections are known to elicit stable populations of IgG + memory s. For example, one study found IgG1 + memory s specific for the 1918 pandemic strain of influenza virus circulating in the blood of survivors 90 years after primary exposure to the virus 50. Thus, it would be interesting to test the possibility of heterogeneity between IgG1 + memory s in terms of their self-renewal and their survival ability, which might result, for example, from differences in antigen, adjuvant and infection route. Robust responsiveness. In T cell-dependent primary responses, it is well known that the production of high-affinity class-switched antibodies requires T FH cells and FDCs. Thus, it is worth considering both -intrinsic and -extrinsic mechanisms to account for the robust responsiveness of memory s. Memory s rapidly differentiate into plasmablasts that produce class-switched antibodies that are capable of clearing the infection far more quickly than naive s this is the basis of vaccination. To explain the rapid response of IgG1 + memory s compared with IgM + naive s, two non-mutually exclusive models have been traditionally postulated (FIG. 2a). In the BCRintrinsic model, the unique IgG1 cytoplasmic domain structure of 28 highly conserved amino acid residues (compared with the IgM cytoplasmic tail, which consists of three amino acids) is thought to be the primary factor accounting for differences in responsiveness, whereas in the BCR-extrinsic model, other changes such as alterations in transcription factor levels that take place during priming are thought to explain the differences. In support of the BCR-intrinsic model, several in vitro biochemical studies have shown differential signalling activity of IgM and IgG1 BCRs. In particular, two binding molecules for the IgG1 cytoplasmic tail were identified. The adaptor protein growth factor receptor-bound protein 2 (GRB2) binds to a conserved phosphorylated tyrosine residue in the cytoplasmic tail of the IgG1 or IgE BCR and thereby enhances proliferation 51. The other molecule is a member of the membrane-associated guanylate kinase (MAGUK) family, DLG1 (also known as SAP97), which is a binding partner of the membrane-proximal region of the cytoplasmic tail of IgG1. DLG1 enhances the accumulation of the IgG1 BCR in the immunological synapse and modulates p38 mitogen-activated protein kinase (MAPK) activity 52. As GRB2 and DLG1 bind to different sites of the IgG1 cytoplasmic tail, they might have a synergistic mode of action. To assess the contribution of the BCR-intrinsic and BCR-extrinsic models, a mouse IgG + naive line was recently established by nuclear transfer from an IgG1 + memory, thus enabling for the first time a direct comparative analysis of naive-type IgG1 + s and antigen-experienced memory-type IgG1 + s (FIG. 2b). Antigen-experienced, but not naive, IgG1 + s rapidly differentiated into plasma cells (FIG. 2c), which indicates that stimulation history (a BCR-extrinsic factor) is important in determining the response 53. Furthermore, the transcription factor BTB and CNC homologue 2 (BACH2), which is known to repress differentiation towards plasma cells, was expressed at a lower level in IgG1 + memory s than in IgG1 + naive s, thus favouring the differentiation of IgG1 + memory s to plasma cells over germinal centre entry. Owing to data showing that before the induction of B lymphocyte-induced maturation protein 1 (BLIMP1; also known as PRDM1) expression (and hence plasma cell differentiation) there exist several intermediate states between activated s and plasma cells (such as a PAX5-downregulated state), we propose that IgG1 + memory s are placed into such an intermediate state by the downregulation of BACH2 (REF. 54). Given that the BACH2 level of IgM + memory s was more similar to that of naive s (in other words, higher than in IgG + memory s), we would also like to propose that IgM + memory s are more similar to naive s in terms of their differentiation state and their ability to enter the germinal centre pathway. The nuclear transfer model 53 shows the importance of stimulation history (the BCR-extrinsic model) for the robust responsiveness of IgG + memory s, but it does not exclude a role for the IgG1 cytoplasmic domain (the BCR-intrinsic model). Indeed, a study using antigen non-experienced s expressing a transgenic hen egg lysozyme (HEL)-specific chimeric IgM IgG1 BCR in which the cytoplasmic domain of the BCR was derived from IgG1 showed that the IgG1 cytoplasmic domain markedly enhanced survival at the plasmablast stage 55. Consistent with this report, but in a physiological setting, IgG-type class-switched plasmablasts survived better than their IgM-type counterparts 56. These studies were carried out during primary responses but this IgG1 dependent mechanism probably functions in recall responses as well. Dependence on other cell types. Although virusspecific memory s can be activated in the absence of T cells 57, T cell help is a strict requirement for the reactivation of memory s that are specific for monomeric protein antigens 32,57. Accumulating evidence has shown that T FH cell-derived, CXC-chemokine receptor 5 (CXCR5)-expressing memory T cells exist in secondary lymphoid tissues or in the circulation (BOX 2) and that they have a crucial role in helping activation These memory T FH cells downregulate expression of most of the canonical markers of effector T FH cells, such as programmed cell death protein 1 (PD1) and BCL 6, but they can upregulate these markers quickly upon restimulation Memory T FH cells maintain low, but detectable, levels of CXCR5 expression, which makes memory T FH cells distinguishable from other types of memory T cell. As a result of this CXCR5 expression, memory T FH cells in the spleen or in the lymph nodes are not only found in the T cell zone but also accumulate 154 MARCH 2015 VOLUME 15

7 a Number of antigen-specific s Naive Class-switched Affinity maturation Memory Antigen-experienced s Long-lasting s BCR-intrinsic model Igβ Igα IgM BCR IgG1 BCR Signal 1 Signal 1 Signal 2 BCR-extrinsic model Transcriptional or epigenetic changes b IgG1 + memory Nuclear transfer Unfertilized egg Antigen exposure Time since antigen exposure (days) Naive Memory Cloned mouse c Transferred cell type IgG1 + naive s CD IgM + naive IgG1 + naive IgG1 + memory Plasma cells B220 Figure 2 Two models to account for the robust responsiveness of memory s. a In the receptor (BCR)-intrinsic model, the differences between BCRs (IgM versus IgG) are primarily responsible for the distinct Nature Reviews Immunology characteristics of the IgM + naive response compared with the IgG + memory response. The highly conserved, unique cytoplasmic domain of IgG-type BCRs, which is thought to recruit additional signalling molecules to the BCR complex (signal 2), endows IgG + s with a more rapid and robust response. In the second model, BCR-extrinsic changes that take place during primary activation, such as alterations in transcriptional networks or epigenetic modifications, are the primary factors that account for the more efficient response of memory s compared with naive s. b A recent study has tested these two models by generating IgG + naive s 53. The cloned mice were established through nuclear transfer from an IgG1 + memory to an unfertilized egg. The cloned mice have IgG1 + s that have never encountered cognate antigen. c IgM + naive, IgG1 + naive or IgG1 + memory s were transferred to antigen-primed recipients and challenged with antigen. Four days later, the differentiation of the transferred cells to plasma cells (B220 low CD138 + cells) was analysed. Antigen-experienced IgG1 + memory s rapidly differentiated into CD138 + plasma cells, whereas IgG1 + naive s behaved similarly to IgM + naive s, which indicates that the IgG cytoplasmic domain alone is insufficient to confer the differentiation activity of IgG + memory s. Part c reprinted from Immunity, 39, Kometani, K. et al., Repression of the transcription factor Bach2 contributes to predisposition of IgG1 memory s toward plasma cell differentiation, (2013), with permission from Elsevier. at the T cell border and in follicles 60,64. Importantly, a recent study showed that loss of memory T FH cells abolished the reactivation of memory s to differentiate into plasma cells 64, which clearly shows the requirement for memory T FH cells in efficient recall antibody responses. Do memory T FH cells contribute to the prompt activation of memory s and, if so, how? Several studies have shown a crucial role for cognate s as antigen-presenting cells. First, deficiency of MHC class II molecules on memory s abolished secondary antibody responses 32,65. Second, the helper activity of human memory T FH cells in peripheral blood was exclusively acquired upon cognate interaction with s but not with dendritic cells 66. Indeed, BCL 6 expression by memory T FH cells was dependent on cognate memory s and not on dendritic cells 64. Given that the upregulation of BCL 6 on T FH cells is induced much more quickly during a recall response than during a primary response, such prompt activation of memory T FH cells NATURE REVIEWS IMMUNOLOGY VOLUME 15 MARCH

8 REVIEWS Box 2 Circulating memory T follicular helper cells The presence of CD4 + T cells expressing CXC-chemokine receptor 5 (CXCR5) in human peripheral blood was first reported in 1994 (REF. 86). These cells were initially thought to be recently activated T cells, but accumulating evidence now indicates that this subset actually contains memory T cells and shares functional properties with effector T follicular helper cells (T FH cells) The relationship between circulating CXCR5 + CD4 + T cells and effector T FH cells has been revealed in studies of samples from patients with a primary immunodeficiency or studies of genetically mutant mice. The development of circulating CXCR5 + CD4 + T cells was found to depend on lymphoma 6 (BCL 6) and inducible T cell co-stimulator (ICOS), but to be independent of signalling lymphocyte activation molecule-associated protein (SAP), which indicates that the circulating CXCR5 + CD4 + memory T cells are predominantly generated from cells committed to the T FH cell lineage but not from mature germinal centre T FH cells Circulating memory T FH cells are a heterogeneous pool of cells expressing various surface molecules. On the basis of their expression of CXCR3 and CC-chemokine receptor 6 (CCR6), circulating memory T FH cells can be broadly categorized into inefficient helpers for s (CXCR3 + CCR6 cells) and efficient helpers for s (CXCR3 CCR6 cells or CXCR3 CCR6 + cells) 88. The differential expression of ICOS, programmed cell death protein 1 (PD1) and CCR7 further defines functionally distinct subpopulations within the subsets 89,91,93. Importantly, an alteration in the balance of these subsets in the blood seems to be associated with continuing humoral responses. For example, individuals infected with HIV who have developed broadly neutralizing antibodies against HIV were found to have a higher frequency of the memory T FH cell subsets that provide efficient help for s than patients with HIV who do not have broadly neutralizing antibodies 89. In a study of the seasonal influenza virus vaccine, an increase in size of the CXCR3 + CCR6 memory T FH cell subset was found at an early time point after vaccination 94. This indicates that the currently used influenza virus vaccine is mainly dependent on memory T FH cells that provide inefficient help for s and this might explain why the vaccine has limited efficacy. Exhaustion A term that was initially used to describe a state of T cell dysfunction that arises during many chronic infections and in cancer, and is typified by the increased expression of programmed cell death protein 1. by cognate memory s, which reside near to each other in the spleen or lymph nodes, is likely to be a crucial determinant for the subsequent rapid activation of memory s (FIG. 3). In addition to memory T cells, FDCs are also thought to contribute to the maintenance of memory s and to subsequent recall responses 44. Although it has been assumed that FDCs retain antigens for extended periods of time, the role of antigen persistence in memory responses is debated and the underlying mechanism of the role of FDCs in memory maintenance is not clear. One theory is that FDCs cycle CR1-bound complement C3d coated immune complexes in non-degrading endosomal compartments, which protects the antigen from degradation, thereby retaining its availability to s for extended periods 67. This mechanism could contribute to germinal centre persistence and to the generation and/or maintenance of memory s. During a recall response, FDCs might contribute by promptly presenting antigens because exogenous protein molecules, particulates and invading pathogens are known to be quickly transported to FDCs, and this transport is accelerated by their binding to pre-existing antibodies and by the subsequent complement activation. As IgG1 + memory s are located near to the contracted germinal centres, which contain FDCs in close proximity, these types of memory s are likely to capture the secondary antigens very quickly 32. Re-diversification. IgM + memory s can reinitiate a germinal centre reaction following antigen reencounter 68. In addition, the persistence of the primary germinal centre seems to be affected by the type of antigen, with particulate antigens being more likely to promote germinal centre survival. Innate receptors also differentially affect the persistence of germinal centre structures; for example, the combination of TLR4 and TLR7 signals strongly enhances germinal centre maintenance, although this study did not address which cell types (dendritic cells, FDCs or s) are responsible for the effect 69. If a pre-existing germinal centre persists as the primary response wanes, s that are newly activated upon recall antigen exposure can efficiently join this primary germinal centre 70. In this situation, when T cell help is shared by already existing germinal centre s and newly joining s, the reuse of the germinal centres is most effective. Although this concept has not yet been directly proven, we think that pre-existing germinal centres probably facilitate re-diversification of affinity-matured BCRs through the recruitment of activated s. Considering the relatively limited capacity of IgG1 + memory s to enter germinal centre reactions, how can IgG1 antibodies of even higher affinity be generated during recall responses? Given that memory T FH cells are required for the activation of IgG1 + memory s, cellular selection probably takes place during the initial interactions between memory T FH cells and IgG1 + memory s at the T cell border. IgG1 + memory s expressing higher-affinity BCRs would be selected to differentiate into plasmablasts in an extra follicular response. Another possibility is that IgM + memory s, through class switching and somatic hyper mutation after entry to the germinal centre, might have a major role in the generation of higher-affinity IgG1 antibodies 9. These two pathways are not mutually exclusive. IgG1 + and IgM + memory s probably participate in the generation of higher-affinity IgG1 antibodies relatively early and late, respectively, after re-encountering antigen. If so, this could be considered as one example of a multi layered immune response; distinct types of memory s that, through their respective functions, complement and reinforce each other over time. Atypical memory s and exhaustion Depending on the antigen, the adjuvant and the infection route, distinct types of memory s can be generated. A unique situation occurs during chronic viral infection, in which there is prolonged and increased antigen load. An important question in this scenario concerns the types of memory s that are generated and whether they can achieve lifelong immunity. However, this issue has not been well studied, with the exception of human HIV infection. Two atypical memory subsets that express low levels of CD21 have been reported in the blood of patients with HIV; namely, CD27 + CD21 low cells (the activated memory subset) and CD27 CD21 low cells (the tissue-like memory subset) 71. The CD27 + CD21 low cell subset expresses higher levels of the activation markers CD80 and CD95 than all other subsets. By contrast, the tissue-like memory subset has been thought to be in a state of exhaustion in vivo for three main reasons. First, these tissue-like memory s can be induced 156 MARCH 2015 VOLUME 15

9 a Naive phase b Primary response follicle BCR Antigen Germinal centre response T FH cell Secondary lymphoid organ T cell border T cell zone Dendritic cell Naive Naive CD4 + T cell Antigen challenge Migration MHC class II Antigen TCR Short-lived plasma cell Migration Antigen-specific IgG concentration Time after antigen challenge (weeks) c Memory phase d Secondary response Secondary germinal centre response Memory Contracted germinal centre Antigen presentation T cell help Memory T FH cell Antigen rechallenge Rapid plasma cell differentiation Antigen-specific IgG concentration Time after antigen challenge (weeks) Figure 3 Regulation of memory activation by memory T follicular helper cells. a Before antigen challenge, the frequency of antigen-specific T cells and s is low and these cells are far apart in secondary lymphoid tissues. b Upon antigen challenge, antigen-specific naive T cells in the T cell zone are first primed by dendritic cells and then migrate towards the follicles. Antigen-specific s that encounter antigen also migrate to the border of the T cell zone, where the cognate interaction between a and a T cell is formed. Some of the activated s differentiate into short-lived plasma cells and others participate in the germinal centre reaction with help from T follicular helper cells (T FH cells). During the primary response, low levels of antigen-specific IgG production are detected around 1 week after antigen challenge (see the inset). c As antigen is cleared, antigen-specific memory T cells and memory s are generated. Some of the memory s reside adjacent to contracted germinal centres. Memory T cells derived from effector T FH cells (memory T FH cells) are localized at T cell borders or in follicles; therefore, memory s and memory T FH cells are in close proximity. d Following antigen rechallenge, memory s function as antigen-presenting cells to efficiently present antigens to cognate memory T FH cells. After obtaining T cell help, the memory s enter rapid plasma cell differentiation and secondary germinal centre formation. In the secondary response, high levels of antigen-specific IgG antibodies are produced within a few days (see the inset). BCR, receptor; TCR, T cell receptor. to differentiate into antibody-secreting cells in vitro, but the corresponding antibodies could not be found in the patients serum. Second, these cells express several inhibitory receptors, including the immuno receptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory receptor Fc receptor-like protein 4 (FCRL4). Third, following small interfering RNA-mediated downregulation of the expression of FCRL4 and sialic acid-binding immunoglobulin-like lectin 6 (Siglec-6), BCR-mediated proliferation of these cells was increased 72. These findings indicate that these inhibitory receptor pathways lead to the exhausted state of the tissue-like memory s and indicate the therapeutic potential of using FCRL4 or Siglec-6 antagonists for reactivating tissue-like memory s, similarly to the reactivation of exhausted CD8 + T cells by PD1 specific monoclonal antibodies 73. A similar increase in the size of the FCRL4 + subset has been observed in other chronic infections (for example, in hepatitis C virus and malaria infections) and in certain autoimmune diseases 74,75. However, in contrast to the case of HIV, a recent malaria study NATURE REVIEWS IMMUNOLOGY VOLUME 15 MARCH

10 REVIEWS shows that the FCRL4 + population does produce protective antibodies, which indicates that chronic antigen exposure during malaria infection induces the accumulation of atypical memory s that can produce broadly neutralizing antibodies, rather than resulting in a dysfunctional memory cell population. Collectively, these studies indicate a need for more intensive research in this area. For example, what is the extent of heterogeneity of these atypical memory subsets? How are these subsets generated to result in their differential functions? How does each type of chronic infection or autoimmune disease affect atypical memory subsets, thereby contributing to disease pathology? Perspective In this Review, we have discussed the recent progress in the field of memory s, particularly focusing on the heterogeneity of memory s and the mechanisms that are thought to contribute to effective recall humoral responses. During reinfection with the same virus, and not a variant virus, memory s seem to function as a simple back up for long-lived plasma cells because longlived plasma cells seem to be more stringently selected in terms of their affinity than memory s 76,77. In this regard, the most crucial properties of memory s may be their longevity and location. This leads to the question of what happens to the response when the reinfection is with a variant virus? Upon reinfection with West Nile virus, longlived plasma cells produced neutralizing antibodies that were specific for a dominant determinant of the original virus but that were poorly reactive with the variant virus, whereas the memory pool contained cells that were capable of producing high-affinity, neutralizing antibodies specific for the variant virus 78. Similarly, memory s that were isolated from humans before the H1N influenza pandemic contained clones with broadly neutralizing properties, which provided the protection against such a pandemic strain of influenza virus 79. Therefore, memory s have a broader repertoire of antigen specificity than long-lived plasma cells, which could provide considerable benefit to host immunity upon infection with a pathogen variant that has not previously been encountered. In this context, several questions need to be answered in the future. Although such cross-reactive (heteroclitic) specificities have been shown to exist in germinal centreexperienced, isotype-switched memory s, are they the only source of such antibodies? Are the germinal centre-independent or IgM + memory populations also involved? This might be the case, as reported in a recent study in which mice immunized with influenza virus in the presence of rapamycin produced mostly IgM antibodies that protected against infection with multiple subtypes of influenza virus 80. Moreover, memory s with lower affinity than germinal centre-dependent cells, such as IgM + or germinal centre-independent memory s, are certainly present at higher frequencies than naive s 10. Those memory s can re-diversify by entering germinal centres upon secondary infection. In this way, the memory compartment might considerably extend the primary antibody repertoire. The molecular features of escape virus variants generated during chronic infection seem to be important for selecting memory s that have broadly neutralizing antibody specificities, which indicates the importance of primary and boosting immunogens 81. 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