REVIEW doi: 10.1111/sji.12336 ..................................................................................................................................................................

Regulation of B Cell to Plasma Cell Transition within the Follicular B Cell Response K.-P. Nera, M. K. Kyla¨niemi & O. Lassila

Abstract Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland

Received 15 May 2015; Accepted in revised form 23 June 2015 Correspondence to: Dr. K.-P. Nera, Department of Medical Microbiology and Immunology, University of Turku, Kiinamyllynkatu 13, 20520 Turku, Finland. e-mail: [email protected]

Persistent humoral immunity depends on the follicular B cell response and on the generation of somatically mutated high-affinity plasma cells and memory B cells. Upon activation by an antigen, cognately activated follicular B cells and follicular T helper (TFH) cells initiate germinal centre (GC) reaction during which high-affinity effector cells are generated. The differentiation of activated follicular B cells into plasma cells and memory B cells is guided by complex selection events, both at the cellular and molecular level. The transition of B cell into a plasma cell during the GC response involves alterations in the microenvironment and developmental state of the cell, which are guided by cell-extrinsic signals. The developmental cell fate decisions in response to these signals are coordinated by cell-intrinsic gene regulatory network functioning at epigenetic, transcriptional and post-transcriptional levels.

Introduction The humoral arm of the adaptive immune system depends on mature B cells which upon activation by an antigen differentiate into antibody secreting plasma cells and memory B cells. It was first realized 50 years ago that B cells constitute a functionally and developmentally distinctive part of the adaptive immune system responsible for humoral immunity [1, 2]. Now, it is general knowledge that at least in the extensively studied immune system of mice there are three main subsets of mature naı¨ve B cells: B1 B cells, marginal zone (MZ) B cells and follicular B cells. Residing predominantly in the peritoneal cavity and at the mucosal sites, B1 cells provide rapid first-line response to the so-called T-independent (TI) antigens, such as bacterial components. MZ B cells carry out a similar function located in the marginal sinus of spleen. While follicular B cells can also respond to TI antigens, they are considered to be specialized responding to antigens that demand help from CD4+ T helper cells. In the lymph nodes, na€ıve follicular B cells are located in B cell follicles, which are surrounded by T cell zones. After encountering exogenous antigen, B cells proliferate and migrate to the border of T cell zone or to the interfollicular region, where they form interactions with antigen-specific CD4+ T cells to become fully activated [3, 4]. A subset of these activated B cells migrates to extrafollicular foci and differentiates into short-lived plasmablasts that secrete low-affinity antibodies, whereas some activated follicular B cells return to the B cell follicle

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to proliferate and form germinal centres (GC). Within GCs, B cells increase their immunoglobulin (Ig) affinity for antigen by a process called affinity maturation, which includes somatic hypermutation (SHM) and subsequent clonal selection, eventually producing long-lived plasma cells homing to the bone marrow, as well as memory B cells. These cell types are responsible for our high-affinity immunological memory and durable antibody responses. Immunoglobulin class switch recombination (CSR) also takes place in GCs. CSR is not, however, restricted to GC microenvironment, as plasmablasts migrating to extrafollicular sites prior to GC formation have often undergone CSR, but not SHM. The terminal differentiation of B cells into antibody secreting cells (ASC), plasmablasts and plasma cells, is a complex process regulated and influenced by many extracellular as well as B cell intrinsic factors. Generation of ASCs that have undergone CSR always involves B cell activation and subsequent proliferation. The regulation of CSR is linked to the cell division cycle itself and appears to be a result of stochastic developmental decisions taken at the single cell level leading to the differentiation of clones with variable alterations, which are proportional to the amount of cell divisions [5–7]. On the other hand, B cell activation and GC formation is a dynamic spatiotemporal process coordinated by B cell intrinsic transcription factors as well as environmental cell-extrinsic signals influencing the cell fate decisions [8–10]. B cell activation and terminal differentiation is controlled by a complex network of transcription factors.

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226 Regulation of B Cell to Plasma Cell Transition K.-P. Nera et al. .................................................................................................................................................................. The most simplistic way to dissect this network is to divide these transcription factors to those promoting B cell phenotype while preventing premature plasma cell differentiation and to those driving the terminal differentiation into ASCs. The key factors sustaining the B cell phenotype are Pax5, Bcl6, Bach2, PU.1 and IRF8, whereas the terminal differentiation is driven by IRF4, Blimp-1 and XBP1. Interestingly, Bcl6, IRF4 and Blimp1 also critically regulate the development and function of follicular T helper (TFH) cells, which provide crucial interactions and signals regulating the GC reaction of B cells [11]. Although we currently have fairly good idea of the basic framework and hierarchy within the core transcriptional network controlling the transition of B cell to plasma cell, we are just starting to understand how it is connected to the regulation at the epigenetic level and how super-enhancers may influence the lineage-specific master regulators within this network. Moreover, posttranscriptional regulation mechanisms by micro-RNAs are involved in fine tuning the expression of critical factors coordinating the core network. In this review, we will discuss the recent advances in our understanding of the regulation of B cell activation, the GC reaction, the terminal differentiation of ASCs, as well as the formation of memory B cells. We aim to elucidate the current understanding of spatiotemporal dynamics in follicular B cell response, and how it is influenced by cell-extrinsic differentiation signals or cell-intrinsic gene regulatory network at transcriptional, epigenetic and post-transcriptional levels.

Dynamics of follicular B cell-mediated humoral response Persistent humoral immunity relies on the production of high-affinity plasma cells and memory B cells, which are generated by the follicular B cell response. Prior to antigen challenge, B cell follicles are populated by IgM+ IgD+ na€ıve B cells. Primary follicular B cell response initiates, when cells from this population recognize an exogenous antigen within the follicle and become activated [3]. Activation is followed by migration to the interfollicular foci, where B cells proliferate and receive help from cognately activated CD4+ helper T cells (Fig. 1), which have adapted TFH phenotype upon activation and subsequently migrated to the foci [12]. This process leads to the full activation of B cells and enables an immediate extrafollicular humoral response as a subset of activated B cells differentiates into short-living plasmablasts [13]. The antibodies secreted during this first-line defence, may have undergone CSR but not SHM, therefore exhibiting only low or moderate affinity for the antigen. However, B cells predominantly differentiating into plasmablasts during the extrafollicular response are the ones with high-affinity antibody specificities within the pool [14, 15].

Few of the activated B cells return to the follicle to form GCs. The mechanism how this population is selected remains unresolved. Nonetheless, the cells with highest relative affinities within the remaining pool are the ones entering the GC program through a process that involves interclonal competition for T cell signals [16–18]. Whereas B cells compete for T cell signals to re-enter the follicle and progress to GC B cell program, TFH cells also depend on signals from B cells in order to fully differentiate into GC TFH cells and for their migration to forming GCs [12]. In fact, dendritic cells initially prime T cells to become TFH cells, prior migration to the interfollicular foci [19], where activated B and T cells both are committed to differentiate into GC B and TFH cells before entering the follicle and start expressing GC-associated proteins [20, 21]. Once mature GC is fully established at day 7 after immunization [10], it has polarized into two microenvironments known as the dark zone and the light zone based on their histological appearance. The dark zone is constituted of densely packed GC B cells, which is aggressively proliferating and undergoing SHM to modify their affinity for the antigen. In contrast, there are clearly less B cells in the light zone, which is also populated by GC TFH cells, follicular dendritic cells (FDC) and macrophages. GC functions as a site for antigen-based affinity maturation, where rapidly proliferating dark zone B cells further diversify their rearranged IgV genes by SHM and produce mutant clones with a variety of affinities for the immunizing antigen. This process is followed by the selection of GC B cells that express the antigen receptors with the highest affinity within the light zone. GC B cells recirculate between the two zones, and repetitive iterative rounds of mutation and selection eventually result in the production of plasma cells and memory B cells that secrete effective high-affinity antibodies. Apparently, TFH cells promote the selection of highaffinity B cells within the light zone [10, 22–24]. Limited amount of intact antigen is constantly presented to GC B cells on the FDCs allowing antigen capture via surface Ig receptor (sIg) and a subsequent presentation of the processed antigen on MHC II to the TFH cells. The affinity of sIg receptor is directly associated with the amount of antigen captured and the density of presented MHC II complexes on the B cell surface [22]. TFH cells form the largest and longest transient contacts with GC B cells presenting the highest densities of antigen on MHC II complexes [25]. As a result, GC B cells with the highest affinity for antigen receive the most TFH cell help and are programmed for enhanced proliferation upon the dark zone re-entry [26], which enhances the affinity maturation further. The fine tuning of selection in the affinity maturation is achieved by the limited amount of antigen presented for GC B cells on FDCs [27]. Antibodies generated in the response earlier enter the GCs freely, masking the antigens that are available for B cells to

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Figure 1 Follicular B cell response. Follicular B cell response is initiated when na€ıve B cells within the lymphoid follicle encounter exogenous antigen and become activated. (1) During the first step of the follicular response, activated B cells migrate to the interfollicular foci to receive help for further activation, proliferation and differentiation from cognately activated CD4+ T helper cells, which have migrated to the foci and started to differentiate into TFH cells. In the interfollicular region, activated B cells present processed antigen to the TFH cells on the MHC class II complex and receive co-stimulation. This B cell T cell communication eventually leads to full activation of follicular B cells. (2) After receiving help from TFH cells, high-affinity B cells within the pool launch immediate first-line defence, extrafollicular response and differentiate to plasmablasts producing low-affinity antibodies for the antigen. (3) Communication between B and T cells within the interfollicular foci also primes a subset of both activated cell types for commitment to GC program and subsequent migration to the centre of B cell follicle where formation of GC is initiated. (4) In the dark zone of established GC, B cells proliferate rapidly while simultaneously undergoing SHM to modify their affinity for the antigen. (5) In the light zone, TFH cells promote the positive selection of highaffinity B cells. Antigens, which are masked by antibodies produced earlier in the response (green), are presented to B cells on FDCs. By competing with existing antibodies for specificity, light zone B cells acquire antigen with their antigen receptor (red) that has been modified in the dark zone through SHM. The acquisition of antigen is directly associated with the affinity and amount of TFH cell help, which is a prerequisite for positive selection that destines B cells to either re-enter the dark zone for further affinity maturation through SHM or exiting GCs. B cells that are not able to compete for antigen or T cell help are deleted through apoptosis. (6) GC reaction produces both plasmablasts and memory B cells. Plasmablasts produced in GC secrete high-affinity antibodies in primary infection. A subset of plasmablasts migrate to a survival niche in the bone marrow where they become long-lived plasma cells, which are maintained by survival signals from reticular cells, eosinophils, megacaryocytes and monocytes. (7) In the secondary infection, high-affinity antibodies produced by plasma cells provide the first-line defence for re-invading pathogen, whereas memory B cells launch an immediate response by differentiation into plasmablasts. Memory B cells may also seed GCs during the secondary immune response and further enhance their affinity for the antigen.

recognize. Thus, only GC B cells expressing surface Igs with high enough affinity to compete with the previously produced antibodies are able to acquire antigens that can be subsequently presented to GC TFH cells in order to receive survival and differentiation signals. This process of antigen masking is also likely to provide means for inter-GC communication, as antibodies can effectively infiltrate to the neighbouring GCs [27]. Moreover, newly activated B cells can reutilize pre-existing GCs [28], and communication between GCs promotes the production of antibody repertoire with the highest possible affinities for the large range of epitopes. In fact, GCs seem to be surprisingly open structures. Follicular B cells from outside the GCs frequently visit the GC compartment suggesting that also they can scan antigens trapped within the GCs [24] and also TFH cells have a capability to exit and re-enter the GCs [12]. Finally, GC B cells that fail to sufficiently compete in

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either antibody recognition and/or for TFH cell help are destined to negative selection through apoptosis. As SHM is a random process, it may potentially lead to selfreactivity. Self-reactive GC B cells can be effectively removed through mechanisms that still remain somewhat obscure. However, under certain circumstances, they can also escape deletion, and the fact that most pathogenic autoantibodies show hallmarks of SHM and selection suggests that failure to achieve self-tolerance in GCs may contribute to many autoimmune diseases [29]. Positively selected light zone GC B cells have three possible fates. It appears that very high-affinity cells have an increased probability to differentiate into plasmablasts, while lower affinity cells can either become memory B cells or recycle back to the dark zone for further rounds of SHM [30]. Once GC B cells possessing the highest affinity for the antigen have been destined to plasmablast fate, they

228 Regulation of B Cell to Plasma Cell Transition K.-P. Nera et al. .................................................................................................................................................................. produce high-affinity antibodies during primary infection as a part of GC response. By a mechanism, which is not yet fully understood, a subset of these plasmablasts migrates through the bloodstream to a survival niche, where sufficient survival signals enable them to become longlived plasma cells [31]. The survival niches for plasma cells are predominantly located in the bone marrow. Plasmablasts are capable of proliferation and migration, but as they have a limited life span, migration to the survival niche has been thought to be a key element in their survival and differentiation into long-lived plasma cells. In the bone marrow survival niche, plasma cells are associated with reticular cells expressing CXCL12 chemokine [32]. In addition, recent evidence suggests that eosinophils, megakaryocytes and monocytes provide crucial cytokines and chemokines for plasma cells within the bone marrow survival niche [33–35]. The cell-extrinsic signals guiding the generation and maintenance of bone marrow plasma cells are intimately linked to the B cell intrinsic preprogrammed potentiation, which appears to be a prerequisite for receiving right extrinsic signals at the right time [31]. Both the B cell extrinsic and intrinsic aspects of B cell to plasma cell transition are discussed later in this review. The formation of memory B cells is one of the least understood aspects of GC reaction. According to the classic definition, memory B cells have undergone SHM [36], and thus are products of the follicular GC response. However, B cell memory for T cell independent antigens that do not involve functional GC formation exists [37]. Moreover, there are memory B cells that have not undergone SHM and/or have arisen independently of GCs [38]. Nonetheless, it appears that the majority of memory B cells are produced in GCs. In terms of number of SHMs and affinity, memory B cells are indistinguishable from GC B cells, but have generally lower affinity for antigen compared with plasma cells [39, 40]. While it is still unclear how B cells are selected to enter the memory cell compartment, it has been suggested that unlike plasma cells, memory B cells differentiate stochastically and simply surviving apoptosis would be sufficient for memory B cell differentiation among positively selected GC B cells [30]. Although many fundamental aspects of memory B cell generation still remain obscure, it is evident that the quick response of memory B cells and their immediate differentiation into plasmablasts following the re-encounter of the antigen is a key element of our humoral immunological memory. Moreover, memory B cells can also remodel their B cell antigen receptors further by entering secondary GCs in reinfection [41], which enhances the durable immunological protection mediated by memory B cells. Generally, memory B cells can be subdivided in two categories, IgM+ and isotype-switched cells, of which IgM+ cells have been suggested to preferentially generate new GCs whereas isotype-switched (IgG+) cells rapidly produce plasmablasts upon the antigen re-encounter [42, 43]. On the other hand,

also IgG+ memory cells are comprised of two subpopulations based on their expression of CD80 and PD-L2. Of these, CD80+PD-L2+ memory B cells differentiate directly to plasmablasts while CD80 PD-L2 cells mostly seed GCs [44]. Thus, the B cell memory apparently consists of several different layers.

Cell-extrinsic microenvironment guiding B cell to plasma cell transition The transition of activated follicular B cells into antibody secreting plasma cells is constantly guided by extrinsic signals: cytokines, chemokines and contacts with coreceptors in the surrounding cells. Eventually, these cellextrinsic signals affect the ongoing transcriptional program within the activated B cells during their differentiation into plasma cells. On the other hand, the cell-intrinsic transcriptional program potentiates the differentiation process at each critical step leading to cell fate decisions. In the follicular B cell response, terminal differentiation to plasma cells can be roughly dissected into three major checkpoints. The first checkpoint involves B cell communication with TFH cells within the interfollicular foci. Cellextrinsic signals from TFH cells at the checkpoint I affect the B cell fate decision enabling them to either launch the extrafollicular response by differentiation into plasmablasts or direct B cells to adapt the GC differentiation program leading to a consequent re-entry to the follicle towards forming GCs. The second major checkpoint takes place in GCs, where communication between GC B and TFH cells within the light zone guides B cell fate decision to either re-enter the dark zone for further SHM or migration out of the GC to become plasmablasts. The third checkpoint involves extrinsic signals for plasmablasts to find their survival niche within the bone marrow, which eventually leads to their differentiation into long-lived plasma cells. Moreover, constant cellular contacts and cell-extrinsic signalling within the bone marrow niche is needed for the maintenance and prolonged survival of plasma cell population. The checkpoints I and II (described above) that eventually lead to the development of follicular GC derived plasma cells both depend on the B cell interactions with TFH cells. Activated T cells destined to TFH cell fate are defined by their high expression of CXCR5 chemokine receptor facilitating their follicular homing after initial priming by DC in the T cell zone [12]. Moreover, TFH cells are defined by the expression of T cell inhibitory receptor PD-1, inducible co-stimulator (ICOS) and IL-21, which are important mediators of communication between B and TFH cells in both checkpoints I and II (Fig. 2A). Most importantly, TFH cell fate is dependent on and defined by the expression of transcriptional repressor Bcl6 upon their initial priming by DCs [45–47]. In addition to TFH cells, Bcl6 expression is crucial for the formation of GC B cells,

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228 Regulation of B Cell to Plasma Cell Transition K.-P. Nera et al. .................................................................................................................................................................. produce high-affinity antibodies during primary infection as a part of GC response. By a mechanism, which is not yet fully understood, a subset of these plasmablasts migrates through the bloodstream to a survival niche, where sufficient survival signals enable them to become longlived plasma cells [31]. The survival niches for plasma cells are predominantly located in the bone marrow. Plasmablasts are capable of proliferation and migration, but as they have a limited life span, migration to the survival niche has been thought to be a key element in their survival and differentiation into long-lived plasma cells. In the bone marrow survival niche, plasma cells are associated with reticular cells expressing CXCL12 chemokine [32]. In addition, recent evidence suggests that eosinophils, megakaryocytes and monocytes provide crucial cytokines and chemokines for plasma cells within the bone marrow survival niche [33–35]. The cell-extrinsic signals guiding the generation and maintenance of bone marrow plasma cells are intimately linked to the B cell intrinsic preprogrammed potentiation, which appears to be a prerequisite for receiving right extrinsic signals at the right time [31]. Both the B cell extrinsic and intrinsic aspects of B cell to plasma cell transition are discussed later in this review. The formation of memory B cells is one of the least understood aspects of GC reaction. According to the classic definition, memory B cells have undergone SHM [36], and thus are products of the follicular GC response. However, B cell memory for T cell independent antigens that do not involve functional GC formation exists [37]. Moreover, there are memory B cells that have not undergone SHM and/or have arisen independently of GCs [38]. Nonetheless, it appears that the majority of memory B cells are produced in GCs. In terms of number of SHMs and affinity, memory B cells are indistinguishable from GC B cells, but have generally lower affinity for antigen compared with plasma cells [39, 40]. While it is still unclear how B cells are selected to enter the memory cell compartment, it has been suggested that unlike plasma cells, memory B cells differentiate stochastically and simply surviving apoptosis would be sufficient for memory B cell differentiation among positively selected GC B cells [30]. Although many fundamental aspects of memory B cell generation still remain obscure, it is evident that the quick response of memory B cells and their immediate differentiation into plasmablasts following the re-encounter of the antigen is a key element of our humoral immunological memory. Moreover, memory B cells can also remodel their B cell antigen receptors further by entering secondary GCs in reinfection [41], which enhances the durable immunological protection mediated by memory B cells. Generally, memory B cells can be subdivided in two categories, IgM+ and isotype-switched cells, of which IgM+ cells have been suggested to preferentially generate new GCs whereas isotype-switched (IgG+) cells rapidly produce plasmablasts upon the antigen re-encounter [42, 43]. On the other hand,

also IgG+ memory cells are comprised of two subpopulations based on their expression of CD80 and PD-L2. Of these, CD80+PD-L2+ memory B cells differentiate directly to plasmablasts while CD80 PD-L2 cells mostly seed GCs [44]. Thus, the B cell memory apparently consists of several different layers.

Cell-extrinsic microenvironment guiding B cell to plasma cell transition The transition of activated follicular B cells into antibody secreting plasma cells is constantly guided by extrinsic signals: cytokines, chemokines and contacts with coreceptors in the surrounding cells. Eventually, these cellextrinsic signals affect the ongoing transcriptional program within the activated B cells during their differentiation into plasma cells. On the other hand, the cell-intrinsic transcriptional program potentiates the differentiation process at each critical step leading to cell fate decisions. In the follicular B cell response, terminal differentiation to plasma cells can be roughly dissected into three major checkpoints. The first checkpoint involves B cell communication with TFH cells within the interfollicular foci. Cellextrinsic signals from TFH cells at the checkpoint I affect the B cell fate decision enabling them to either launch the extrafollicular response by differentiation into plasmablasts or direct B cells to adapt the GC differentiation program leading to a consequent re-entry to the follicle towards forming GCs. The second major checkpoint takes place in GCs, where communication between GC B and TFH cells within the light zone guides B cell fate decision to either re-enter the dark zone for further SHM or migration out of the GC to become plasmablasts. The third checkpoint involves extrinsic signals for plasmablasts to find their survival niche within the bone marrow, which eventually leads to their differentiation into long-lived plasma cells. Moreover, constant cellular contacts and cell-extrinsic signalling within the bone marrow niche is needed for the maintenance and prolonged survival of plasma cell population. The checkpoints I and II (described above) that eventually lead to the development of follicular GC derived plasma cells both depend on the B cell interactions with TFH cells. Activated T cells destined to TFH cell fate are defined by their high expression of CXCR5 chemokine receptor facilitating their follicular homing after initial priming by DC in the T cell zone [12]. Moreover, TFH cells are defined by the expression of T cell inhibitory receptor PD-1, inducible co-stimulator (ICOS) and IL-21, which are important mediators of communication between B and TFH cells in both checkpoints I and II (Fig. 2A). Most importantly, TFH cell fate is dependent on and defined by the expression of transcriptional repressor Bcl6 upon their initial priming by DCs [45–47]. In addition to TFH cells, Bcl6 expression is crucial for the formation of GC B cells,

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230 Regulation of B Cell to Plasma Cell Transition K.-P. Nera et al. .................................................................................................................................................................. mutations in either CD40 or CD40L lead to a severely impaired B cell CSR and to the development of Hyper-IgM syndrome (HIGM) [65]. Moreover, patients with CD40L deficiency are characterized with diminished populations of memory B cells [66, 67], which have aberrantly undergone SHM events [67]. GC B cells are by nature pro-apoptotic and depend on survival signals, such as CD40-CD40L ligation and IL-21, provided by GC TFH cells. In addition, PD-1 expressed highly on GC TFH cells appears to enhance the survival of GC B cells and shape the outcome of GC reaction. Like the expression of CD40L, the PD-1 expression is induced on the surface of activated T cells by extended TCR signalling [68, 69] and a high expression of PD-1 on GC TFH cells is likely a consequence of repeated T to B cell interactions that are involved in GC reaction. Whereas the inhibitory receptor PD-1 apparently inhibits the excess proliferation of activated T cells upon TCR cross-linking, both PD-1 and PD-1L (expressed by GC B cells) deficiencies in mice have been shown to result in increased apoptosis of GC B cells and reduced numbers of long-lived plasma cells [70]. The interzonal circulation of GC B cells between the dark and light zones within GCs is guided by their expression of chemokine receptors CXCR4 and CXCR5 [71, 72] as well as activation markers CD83 and CD86 [22]. The dark zone B cells exhibit a CXCR4hi, CD83lo, CD86lo phenotype whereas the light zone B cells are characterized by a CXCR4lo, CD83hi, CD86hi phenotype. CXCR4-deficient GC B cells are retained in the light zone and have generally undergone less SHM events than normal cells [73]. Interestingly, while the upregulation of CXCR4 expression seems to be a prerequisite for GC B cells for the light zone exit and eventual SHM by recirculation to the dark zone, it also appears to have a critical role in the exit of plasmablasts from the GCs [31]. The exact mechanisms how plasmablasts exit GCs or how the cells becoming plasma cells are homing to the bone marrow survival niche (Fig. 2B) still remain to be precisely determined. However, chemokine receptor CXCR4 is believed to be involved in the process and almost all IgG+ plasma cells within the bone marrow are predominantly in close contact with CXCL12 (ligand for CXCR4) expressing reticular cells [32]. The retention and maturation of plasma cell precursors within the bone marrow seem to be further facilitated by interaction between very late antigen 4 (VLA4) with VCAM1 [74]. The survival of long-lived plasma cells also critically depends on their BCMA receptor and APRIL signalling [75, 76]. Eosinophils are the highest producers of APRIL within the bone marrow [33], and similar to plasma cells they express the CXCR4, which possibly facilitates their migration to the close proximity of plasma cells [33, 77]. Eosinophils are currently considered as one of the most potent survival-signal providers for plasma cells within the bone marrow. Other prominent cell types to provide bone marrow survival signals, such as APRIL and

IL-6, for plasma cells are monocytes and megakaryocytes [34, 35].

Cell-intrinsic gene regulatory network potentiating B cell to plasma cell transition The differentiation of activated B cell into a plasma cell involves constant alterations in the microenvironment and developmental state of the cell, which are guided by cellextrinsic signals. The developmental cell fate decisions taken in response to these signals, however, are ultimately based on and restricted by the ongoing cell-intrinsic transcriptional program. At transcriptional level, the differentiation of activated B cells into plasma cells requires coordinated changes in the expression of hundreds of genes, as the transcriptomes of B cells and plasma cells are remarkably different [78]. Still the transition from B cell to plasma cell program appears to be coordinated by a highly hierarchic network of fairly few core transcription factors (Of note: not all of them are discussed in this review). The key factors within this network are functionally antagonistic, as factors including Pax5, Bcl6, Bach2 and IRF8 function to promote the B cell phenotype, whereas the main function of IRF4, Blimp1 and Xbp1 is to initiate the transcriptional program of plasma cells. Once the B cells are positively selected in the light zone of GCs (checkpoint II), they start to gradually change their B cell transcriptome towards the one of plasma cells. This is driven by a coordinate downregulation of transcription factors promoting B cell program and by an upregulation of transcription factors supporting the plasma cell differentiation (Fig. 3A). The downregulation of B cell-specific transcription factor Pax5 appears to be one of the initial events in this process [79, 80]. Being an absolute prerequisite for both the B cell lineage commitment as well as the maintenance of B cell identity, Pax5 is expressed throughout the B cell lineage up until the initiation of ASC differentiation [81]. Within the B cell lineage, Pax5 functions to suppress the expression of lineage inappropriate genes while promoting the expression of B cell lineage genes. These genes are encoding several key components of BCR and its signalling pathway, as well as several key transcription factors regulating the GC reaction, including Bach2, IRF4 and IRF8 [82, 83]. Of these factors, Bach2 and IRF8 promote the B cell program by suppressing the Blimp1 expression [84–86]. IRF4 functions in a dose-dependent manner [87], with low IRF4 levels promoting the GC fate by activating the expression of Bcl6 (Fig. 3B). In contrast, high amounts of IRF4 repress the Bcl6 expression and activate the expression of Blimp1 and ZBTB20 promoting the plasma cell fate [87– 89]. Thus, IRF4 appears to have a dual role in the regulation of B cell to plasma cell transition, as depending on its expression level, it can promote both programs. This kinetic control is associated with concentration-dependent

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K.-P. Nera et al. Regulation of B Cell to Plasma Cell Transition 231 .................................................................................................................................................................. A

B Figure 3 Transcription factors regulating B cell to plasma cell transition. (A) Developmental stages during follicular B cell differentiation into plasma cells and the expression of key transcription factors regulating the process. (B) Gene regulatory network coordinating B cell to plasma cell transition depicted in both B cell and ASC. Transcription factors promoting the B cell fate are highlighted with green and plasma cell promoting transcription factors with red. Activation is marked with red arrows and inhibition by blue lines. Dotted lines show the regulatory mechanisms downregulated during B cell to plasma cell transition.

differential binding abilities of IRF4, as lower amounts of IRF4 co-operatively binds as IRF4-PU.1 or IRF4-BATF complex to the Ets or AP-1 motifs to regulate B cellrelated genes, whereas the regulation of plasma cell program is mediated by binding to the interferon sequence response motifs [88]. Moreover, IRF8-PU.1 complexes are capable of binding to the same regulatory motifs than IRF4-PU.1 complexes limiting their accessibility for IRF4-mediated regulation. IRF8-PU.1 complexes promote the expression of Pax5 and Bcl6 by this mechanism, while simultaneously inhibiting Blimp1 expression [86]. Whereas Pax5 is responsible for maintaining the B cell identity itself during the GC reaction, a common function of Bcl6, Bach2 and IRF4 (low amount) appears to be more related to the maintenance of GC reaction. Bcl6 can be considered as a master regulator of GC reaction, as it is indispensable for the formation of both GC B and TFH cells, and therefore, for the formation of GC itself. Within GCs, Bcl6 functions to prevent DNA damage response during the proliferation in the dark zone [49], thus allowing successful SHM. In addition, Bach2 and IRF4 contribute to the regulation of SHM as well as CSR, as they are known to activate the expression of AID (Activation induced cytidine-deaminase, encoded by aicda). In addition, Bcl6

Ó 2015 The Foundation for the Scandinavian Journal of Immunology

has also been proposed to suppress Blimp1 expression [90], to which it may further contribute by activating the expression of Bach2 [91], a known suppressor of Blimp1 [92]. Pax5, in turn, is thought to suppress the expression of XBP-1 [93], which functions to regulate genes involved in the unfolded protein response (UPR), a signalling pathway activated by the stress in endoplasmic reticulum (ER) by the accumulation of unfolded proteins. UPR is essential in highly secretory cells, such as plasma cells. Blimp1 is considered as a master regulator of plasma cell program, as its expression is essential for the formation of mature plasma cells [94], and it represses the key regulators of B cell program including Pax5 and Bcl6. However, it appears that Blimp-1 upregulation is not the initiative event in the B cell to plasma cell transition, and downregulation of Pax5 and Bcl6 begins before the induction of Blimp1 expression in ASC differentiation [80]. Recent evidence suggests that in addition to the transcriptional level, some of the factors within the gene regulatory network described above (Fig. 3), can be regulated at epigenetic, post-transcriptional or mitochondrial level as well. Pax5 binds to more than 8000 genes, but appears to regulate only a small proportion of them. More importantly, genes regulated by Pax5 during the

230 Regulation of B Cell to Plasma Cell Transition K.-P. Nera et al. .................................................................................................................................................................. mutations in either CD40 or CD40L lead to a severely impaired B cell CSR and to the development of Hyper-IgM syndrome (HIGM) [65]. Moreover, patients with CD40L deficiency are characterized with diminished populations of memory B cells [66, 67], which have aberrantly undergone SHM events [67]. GC B cells are by nature pro-apoptotic and depend on survival signals, such as CD40-CD40L ligation and IL-21, provided by GC TFH cells. In addition, PD-1 expressed highly on GC TFH cells appears to enhance the survival of GC B cells and shape the outcome of GC reaction. Like the expression of CD40L, the PD-1 expression is induced on the surface of activated T cells by extended TCR signalling [68, 69] and a high expression of PD-1 on GC TFH cells is likely a consequence of repeated T to B cell interactions that are involved in GC reaction. Whereas the inhibitory receptor PD-1 apparently inhibits the excess proliferation of activated T cells upon TCR cross-linking, both PD-1 and PD-1L (expressed by GC B cells) deficiencies in mice have been shown to result in increased apoptosis of GC B cells and reduced numbers of long-lived plasma cells [70]. The interzonal circulation of GC B cells between the dark and light zones within GCs is guided by their expression of chemokine receptors CXCR4 and CXCR5 [71, 72] as well as activation markers CD83 and CD86 [22]. The dark zone B cells exhibit a CXCR4hi, CD83lo, CD86lo phenotype whereas the light zone B cells are characterized by a CXCR4lo, CD83hi, CD86hi phenotype. CXCR4-deficient GC B cells are retained in the light zone and have generally undergone less SHM events than normal cells [73]. Interestingly, while the upregulation of CXCR4 expression seems to be a prerequisite for GC B cells for the light zone exit and eventual SHM by recirculation to the dark zone, it also appears to have a critical role in the exit of plasmablasts from the GCs [31]. The exact mechanisms how plasmablasts exit GCs or how the cells becoming plasma cells are homing to the bone marrow survival niche (Fig. 2B) still remain to be precisely determined. However, chemokine receptor CXCR4 is believed to be involved in the process and almost all IgG+ plasma cells within the bone marrow are predominantly in close contact with CXCL12 (ligand for CXCR4) expressing reticular cells [32]. The retention and maturation of plasma cell precursors within the bone marrow seem to be further facilitated by interaction between very late antigen 4 (VLA4) with VCAM1 [74]. The survival of long-lived plasma cells also critically depends on their BCMA receptor and APRIL signalling [75, 76]. Eosinophils are the highest producers of APRIL within the bone marrow [33], and similar to plasma cells they express the CXCR4, which possibly facilitates their migration to the close proximity of plasma cells [33, 77]. Eosinophils are currently considered as one of the most potent survival-signal providers for plasma cells within the bone marrow. Other prominent cell types to provide bone marrow survival signals, such as APRIL and

IL-6, for plasma cells are monocytes and megakaryocytes [34, 35].

Cell-intrinsic gene regulatory network potentiating B cell to plasma cell transition The differentiation of activated B cell into a plasma cell involves constant alterations in the microenvironment and developmental state of the cell, which are guided by cellextrinsic signals. The developmental cell fate decisions taken in response to these signals, however, are ultimately based on and restricted by the ongoing cell-intrinsic transcriptional program. At transcriptional level, the differentiation of activated B cells into plasma cells requires coordinated changes in the expression of hundreds of genes, as the transcriptomes of B cells and plasma cells are remarkably different [78]. Still the transition from B cell to plasma cell program appears to be coordinated by a highly hierarchic network of fairly few core transcription factors (Of note: not all of them are discussed in this review). The key factors within this network are functionally antagonistic, as factors including Pax5, Bcl6, Bach2 and IRF8 function to promote the B cell phenotype, whereas the main function of IRF4, Blimp1 and Xbp1 is to initiate the transcriptional program of plasma cells. Once the B cells are positively selected in the light zone of GCs (checkpoint II), they start to gradually change their B cell transcriptome towards the one of plasma cells. This is driven by a coordinate downregulation of transcription factors promoting B cell program and by an upregulation of transcription factors supporting the plasma cell differentiation (Fig. 3A). The downregulation of B cell-specific transcription factor Pax5 appears to be one of the initial events in this process [79, 80]. Being an absolute prerequisite for both the B cell lineage commitment as well as the maintenance of B cell identity, Pax5 is expressed throughout the B cell lineage up until the initiation of ASC differentiation [81]. Within the B cell lineage, Pax5 functions to suppress the expression of lineage inappropriate genes while promoting the expression of B cell lineage genes. These genes are encoding several key components of BCR and its signalling pathway, as well as several key transcription factors regulating the GC reaction, including Bach2, IRF4 and IRF8 [82, 83]. Of these factors, Bach2 and IRF8 promote the B cell program by suppressing the Blimp1 expression [84–86]. IRF4 functions in a dose-dependent manner [87], with low IRF4 levels promoting the GC fate by activating the expression of Bcl6 (Fig. 3B). In contrast, high amounts of IRF4 repress the Bcl6 expression and activate the expression of Blimp1 and ZBTB20 promoting the plasma cell fate [87– 89]. Thus, IRF4 appears to have a dual role in the regulation of B cell to plasma cell transition, as depending on its expression level, it can promote both programs. This kinetic control is associated with concentration-dependent

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