The genetic network controlling plasma cell differentiation

Seminars in Immunology 23 (2011) 341–349

Contents lists available at SciVerse ScienceDirect

Seminars in Immunology journal homepage: www.elsevier.com/locate/ysmim

Review

The genetic network controlling plasma cell differentiation Stephen L. Nutt a,b,∗ , Nadine Taubenheim a,b , Jhagvaral Hasbold a,b , Lynn M. Corcoran a,b , Philip D. Hodgkin a,b,∗ a b

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3050, Australia Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia

a r t i c l e

i n f o

Keywords: Plasma cell B cell Transcription factor Gene regulatory network

a b s t r a c t Upon activation by antigen, mature B cells undergo immunoglobulin class switch recombination and differentiate into antibody-secreting plasma cells, the endpoint of the B cell developmental lineage. Careful quantitation of these processes, which are stochastic, independent and strongly linked to the division history of the cell, has revealed that populations of B cells behave in a highly predictable manner. Considerable progress has also been made in the last few years in understanding the gene regulatory network that controls the B cell to plasma cell transition. The mutually exclusive transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors, those that maintain the B cell program, including Pax5, Bach2 and Bcl6, and those that promote and facilitate plasma cell differentiation, notably Irf4, Blimp1 and Xbp1. In this review, we discuss progress in the definition of both the transcriptional and cellular events occurring during late B cell differentiation, as integrating these two approaches is crucial to defining a regulatory network that faithfully reflects the stochastic features and complexity of the humoral immune response. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Terminal differentiation is a generally irreversible process that, in the haematopoietic system, leads to the acquisition of specialized effector functions and exit from the cell cycle. The terminal differentiation process is also associated with profound alterations in the morphology, lifespan and gene expression profiles of the differentiated cells compared to their predecessors. The transition of B cells into antibody-secreting cells (ASCs) represents one such terminal differentiation process that is essential for the adaptive immune response. There are three main subsets of mature B cells; follicular B cells, which represent the majority of naïve B cells, marginal zone (MZ) and B1 B cells. MZ B cells reside in the marginal sinus of the spleen, whereas B1 B cells occur predominantly in the peritoneal cavity and at mucosal sites [1]. In contrast, conventional or follicular B cells are almost exclusively found in the lymphoid follicles of the spleen and lymph nodes. The relative propensity of B cells to undergo terminal differentiation varies between the subsets, with MZ and B1 B cells

∗ Corresponding authors at: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3050, Australia. Tel.: +61 3 9345 2483; fax: +61 3 9347 0852. E-mail addresses: [email protected] (S.L. Nutt), [email protected] (P.D. Hodgkin). 1044-5323/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2011.08.010

being specialized, both geographically and genetically, for rapid response to so-called T-independent antigens (TI), such as bacterial components [2]. Follicular B cells may also respond to TI antigens, but appear specialized for responding to antigens that also elicit CD4+ helper T cell responses. Upon antigen encounter and receipt of T cell help, follicular B cells undergo multiple rounds of division and have the unique ability to differentiate into both ASCs (plasmablasts and plasma cells) and memory B cells [1]. Plasmablasts are short-lived, cycling ASCs that are found in extrafollicular foci in peripheral lymphoid organs. Plasmablasts have often undergone immunoglobulin class switch recombination (CSR), but not somatic hypermutation (SHM) to increase the affinity of the resulting antibodies for the antigen. In a poorly understood process, some activated B cells return to the B cell follicle and proliferate vigorously to form a germinal centre (GC), where the B cells initiate SHM to enable further clonal selection. The GC produces long-lived plasma cells that are non-cycling and home preferentially to the bone marrow [1]. The GC reaction also produces memory B cells. These cells maintain a B cell phenotype but can rapidly differentiate into ASCs following re-exposure to antigen. The process of B cell terminal differentiation can be studied in vitro, as B cells are capable of both CSR and ASC differentiation in response to T-cell derived stimuli (CD40 ligation and cytokines) or TI-related signals (Toll-like receptor (TLR) ligation by pathogen derived products such as lipopolysaccharide (LPS) and hyper-methylated CpG DNA). B cell responses in vitro thus provide

342

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

Fig. 1. Division-linked B cell differentiation. (A) Division tracking by dye dilution (cell trace violet, CTV) reveals that switching to IgG1 expression (upper panel) and differentiation to Blimp1-GFP+ ASCs (lower panel) occurs in a proportion of activated B cells in late rounds of cell division. (B) Illustration of how division-linked differentiation is influenced by extrinsic factors. Altering the IL4 concentration regulates the frequency of B cell isotype-switching (to IgG1) and differentiation (to express Syndecan1+ , a marker of ASCs) per cell division. (C) Division number can be used as a reference for assessing the effect of genetic mutations. The upper panel shows Syndecan1 expression on wild-type cells, while the lower panel shows that the development of ASCs in Obf1-deficient mice is accelerated on a per division basis, but leads to incomplete Syndecan1 induction [45].

a controlled system to investigate the biology of B cell terminal differentiation on both a cellular and molecular level. Quantitative analysis of in vitro B cell cultures has revealed a striking relationship between cell division history and CSR and ASC differentiation [3–5]. The proportion of B cells that undergo either of these differentiation events typically increases with each consecutive division (Fig. 1). The time taken to traverse each cell cycle is highly variable between cells, but does not seem to alter the differentiation rate, implying that cell division itself is playing a critical role in tuning the molecular machinery controlling CSR and the generation of ASCs. Furthermore, T cell cytokines such as IL4 and IL5 alter the probability of CSR and ASC differentiation with division (Fig. 1B). Hasbold et al. found that CSR and development of ASCs behaved as independent stochastic processes, allowing the numbers of class switched ASCs in each division to be predicted for different cytokine concentrations and combinations [5]. These findings have lead to a division-based model of B cell behaviour that describes how stochastic decisions taken at a single cell level result in the controlled generation of a variety of differentiated cell types in the population as a whole [5]. Complementing these cellular studies, the last few years have seen major advances in our understanding of the transcriptional regulation of the B cell to ASC transition [6,7]. The gene expression changes that are required for this process are regulated by the coordinated activity of a small group of so-called master regulatory transcription factors. These factors can be divided into those, such as Pax5 and Bcl6, which promote and maintain the B cell program, and others such as Blimp1 and Irf4 that control ASC differentiation. The B cell and ASC factors appear to regulate mutually antagonistic transcriptional programs resulting in a gene regulatory network that ensures the separation of the B cell and ASC fates [6]. While most studies to date have proposed that these master regulatory transcription factors function in a dominant manner to either maintain the B cell fate or to drive ASC differentiation, such deterministic behaviour has not yet been reconciled with the evidence supporting stochastic, division-based regulation of differentiation outlined above. In this review we will discuss both the transcriptional and cellular models of B cell terminal differentiation to draw attention to the need for a model of the genetic network of ASC differentiation that more accurately describes the flexible, dynamic and complex regulation of the adaptive immune response.

2. Factors that promote the B cell fate One class of transcription factors can be conveniently grouped into those whose primary function is to promote the B cell gene expression program and to prevent ASC differentiation. Here we have focused on the best-characterized B cell factors: Pax5, Bcl6, Bach2 and Oct2/Obf1. It should be noted, however, that a number of other transcriptional regulators, including PU.1 [8], Irf8 [9], MITF [10] and Ets1 [11] are also thought to play roles in the process (Fig. 2). 2.1. Pax5 Pax5 functions as the master regulator of the B cell program [12]. Pax5 expression is induced at the onset of B cell differentiation, at the pre-pro-B cell stages, and its expression is maintained at a relatively constant level in all developmental stages and subsets of B cells [13]. Pax5 is required for the initial commitment of lymphoid progenitors to the B cell lineage, as Pax5-deficient pro-B cells maintain a multilineage potential [14]. Remarkably this maintenance of B-cell identity by Pax5 extends throughout the lineage, as conditional inactivation of Pax5 in mature B cells results in the loss of B cell identity [15]. These studies unequivocally demonstrate that B-cell commitment is not a discrete event but is continually maintained by Pax5 throughout the life of a B cell. Pax5 expression is finally silenced upon commitment to the ASC fate [16]. Pax5 controls B cell identity in mature B cells through the simultaneous activation of a large number of B cell associated genes, including those encoding components of the B cell receptor, such as IgH, Blnk, and Ig␣, immune receptors such as CD19, CD21, CD23 and important transcription factors including Irf4, Irf8, Bach2 (Fig. 3A) and Aiolos [17,18]. Pax5 also represses the transcription of an array of genes encoding proteins not usually associated with B cells or ASC, including Flt3, Notch1, M-CSFR, CCR2 and CD28 [17,19–21]. The re-expression of these Pax5-repressed genes also occurs during the physiological down-regulation of Pax5 in ASCs [18]. Interestingly, the conditional deletion of Pax5 in mature B cells [18] or in the chicken DT40 B cell line [22] also results in premature expression of genes encoding proteins involved in plasma cell differentiation and function, including Xbp1, Blimp1, J chain and secreted immunoglobulin. Together these studies suggest that a key function of Pax5 in mature B cells is to repress the plasma cell pathway.

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

343

Fig. 2. The cellular stages and expression pattern of important transcriptional regulators of late B cell differentiation. Naïve B cells are quiescent and express a unique suite of transcription factors. Upon antigen exposure in vivo, or activation with either T cell derived factors (CD40L and cytokines) or TLR ligands in vitro, naïve B cells are induced to enter cell cycle to become an activated B cell. Here activated B cells are divided into an early phase that directly follows the initial mitogenic signal and a late phase that follows in subsequent cell cycles. Some transcription factors, including Obf1 and Irf8 are induced by activation, whereas Irf4 is induced to an intermediate amount in activated B cells. Upon initiation of the terminal differentiation program, activated B cells decrease expression of the indicated B cell-associated transcription factors and further increase Irf4 expression to drive differentiation into ASCs. ASCs can be divided into cycling short-lived plasmablasts and post-mitotic, often long-lived plasma cells based on the expression of Blimp1.

2.2. Bcl6 Bcl6 is a transcriptional repressor that has been extensively studied in the context of its oncogenic role in human B cell lymphoma [23]. While Bcl6 plays some role in bone marrow B cell development [24], it is very highly expressed in GC B cells [25] and is essential for their formation [26–28]. Bcl6 is required during the extensive proliferation that characterizes GC B cells and to allow SHM, through its function as an inhibitor of the DNA

damage response [23]. Conversely, constitutive Bcl6 expression in mice results in expanded GC and ultimately lymphoma [29]. In keeping with this broad function in GC B cells, Bcl6 binds to the regulatory regions of many thousands of genes in GC including cell cycle inhibitor, Cdkn1a (encoding p21), Tp53 (encoding p53) and oncogenes such as Myc and Bcl2 [30,31]. One other proposed target of Bcl6-mediated repression is Blimp1, thereby inhibiting ASC differentiation in the GC [32]. The regulation of the Bcl6 gene is complex, with its mRNA being transcribed in many lymphoid

Fig. 3. Gene regulatory network controlling the transition of B cells to antibody secreting cells. (A) Schematic of the published interactions of the indicated transcription factors with the regulatory regions of the same genes in B cells compared to plasma cells. Horizontal arrows indicate the transcription initiation site. Note the exon/intron structures and position of regulatory regions are schematic only and not intended to be accurate representations. Transcription factor binding sites have been identified in the promoters (upstream of the transcription initiation site) and in regulatory elements in introns and downstream of the coding sequences (again not to scale). Red crosses indicate transcriptional repression, ?? indicate unknown mechanisms. (B) Model of the transcriptional network controlling B cell terminal differentiation as proposed by Staudt [47] and Calame [84]. This model is centred on the transcriptional repression between the B cell promoting factors, Pax5, Bcl6, Mitf and Bach2 (in blue) and the ASC promoting factors Xbp1, Blimp1 and Irf4 (green) that ensures the mutually exclusive nature of the two gene expression programs. ↑, Indicates positive influence on gene expression or function, , indicates repressive activity.

344

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

cells, but the Bcl6 protein is largely restricted to GC B cells through several post-translational mechanisms [23]. The cytokine IL-21 [33,34] and the transcription factor Irf8 [9] are thought to sustain Bcl6 expression in GC, while Irf4 [35] and Blimp1 [36] are proposed to repress Bcl6. Whereas Bcl6 is critical for GC B cells, it appears to play a lesser role in the differentiation of other B cell subsets and during in vitro differentiation of ASCs [37]. 2.3. Bach2 Bach2 is a transcriptional repressor that is expressed throughout B cell differentiation [38], under the positive control of Pax5 [18], before being down-regulated in ASCs. Bach2-deficient mice have strongly impaired GC formation and lack CSR, SHM and Aicda (encoding Activation induced cytidine deaminase (AID)) expression [39]. One crucial target of Bach2 in B cells is Blimp1 [40]. In the absence of Bach2, Blimp1 is prematurely expressed and ASC differentiation is enhanced ([41], Fig. 3). As Blimp1 silences Aicda, premature Blimp1 expression may explain the Bach2-deficient phenotype both in terms of increased ASC differentiation and reduced CSR/SHM. This hypothesis was recently confirmed by experiments that showed that CSR could be rescued in Bach2−/− B cells by inactivating Blimp1 [41]. Thus Bach2 is a crucial component of the genetic network controlling the timing of ASC differentiation. 2.4. Oct2/Obf1 The octamer binding factor, Oct2 and its co-activator, Obf1 are critical regulators of the GC response, as well as MZ and B1 B cell development [42–44]. In vitro differentiation experiments revealed that Obf1 plays two contrasting roles in the development of ASCs [45]. In response to T cell dependent stimulation, Obf1−/− B cells prematurely upregulate Syndecan1 (CD138), a marker of ASCs, as well as Blimp1, suggesting a premature commitment to the terminal differentiation pathway. However, the mutant B cells are unable to complete this process and generate ASCs (Fig. 1C). This function of Obf1 is restricted to T cell dependent cytokine stimulation, as Obf1-deficient B cells respond normally in vitro to the TLR4 ligand LPS [45]. This differential reliance of T cell dependent antibody responses on Obf1 was also observed in vivo in response to immunisation [45]. Thus Obf1, by acting as a molecular brake, is essential for the division-linked differentiation of ASCs (Fig. 1C). Oct2, in contrast, is required more specifically for the IL5-mediated induction of Blimp1 and ASC differentiation, through direct activation of the gene encoding the IL-5R␣ chain [46]. Thus Oct 2 and Obf1 have both common and independent functions during B cell terminal differentiation.

high amounts appear to repress Bcl6 and activate Blimp1 and thus promote the ASC fate [53]. The direct regulation of Blimp1 by Irf4, while initially controversial [53,54], has now been confirmed in the context of IL-21 stimulation, where IL-21 induced phospho-STAT3 binds to a regulatory region of the Blimp1 gene in a complex that contains and requires Irf4 [55]. Thus Irf4 is a pivotal player in the transition to the ASC stage. Two other members of the Irf family are known to be involved in B cell differentiation. Irf8 is the closest relative of Irf4 and is expressed very highly in GC B cells. Irf8 was initially proposed to play a role in GC formation, principally through the promotion of Bcl6 and Aicda expression [9] and to act redundantly with Irf4 to regulate Ig recombination in pre-B cells [56]. However the recent development of a B-cell specific knockout has shown that Irf8 acts predominantly to restrict the size of the follicular and MZ B cell compartments [57]. Irf5, which is more closely related to Irf3, is involved in the type I IFN and inflammatory responses [58,59]. Interestingly, Irf5−/− mice have decreased ASC numbers, a phenotype that was ascribed to the direct regulation of Blimp1 by Irf5 [60]. 3.2. Blimp1 Blimp1, encoded by the Prdm1 gene, is thought to be predominantly a transcriptional repressor that is essential for the terminal differentiation of B and T cells [61], while playing less well-defined roles in other hematopoietic lineages such as dendritic cells [62], macrophages [63] and natural killer cells [64,65]. Within the B cell lineage Blimp1 is expressed in all ASCs, with cycling plasmablasts being distinguishable from long-lived plasma cells based on their expression of Blimp1 in both mouse ([16], Fig. 2) and human [66]. Blimp1 is not essential for the initiation of the ASC program, as a pre-plasmablast population can be generated in its absence [67]. Blimp1 is however required for the further differentiation into fully functional ASCs both in culture and in vivo [68]. Blimp1 has been proposed to have many functions in ASCs including repression of regulators and features of the B cell program, including the genes encoding Spi-B, Id3, CIITA, Myc and notably Pax5 ([69–72], Fig. 3). Blimp1 is also proposed to directly repress Bcl6 in both T and B cells [36]. Interestingly, the consensus Blimp1 binding site is virtually identical to the site bound by Irf1 and Irf2, suggesting that competition for DNA binding may occur between these positive and negative regulatory factors on some gene regulatory sequences [73,74]. These studies clearly demonstrate that Blimp1 is a major executor of the ASC program, and indicate that important mechanistic relationships still need further investigation. 3.3. Xbp1

3. Factors that promote the antibody secreting cell fate 3.1. Irf4 Irf4 is a multi-functional transcriptional regulator that controls many aspects of B cell differentiation including Ig gene recombination, CSR, GC B cell formation and ASC differentiation [47]. Irf4 also is broadly required for the differentiation of CD4+ T cells [48]. Irf4 can bind to DNA weakly on its own, but displays strong co-operative binding in the presence of PU.1, or the closely related Spi-B [49,50]. Irf4-PU.1 dimers have been shown to be important in the regulation of the Igh and Ig loci as well as binding the B-cell specific enhancer of Pax5 [49,51,52]. In mature B cells Irf4 expression is maintained at a low level by the transcription factor, Mitf, as Mitf-deficient B cells undergo spontaneous differentiation to ASCs ([10], Figs. 2 and 3). Importantly, Irf4 appears to function as a dose dependent rheostat, with relatively lower concentrations of Irf4 promoting GC fate and CSR, potentially through the direct activation of Aicda, while

The mammalian unfolded protein response (UPR) is a signalling pathway that responds to endoplasmic reticulum (ER) stress induced by the accumulation of unfolded proteins [75]. Controlling the UPR is essential for highly secretory cell types such as ASCs. One consequence of ER stress is the activation of the transcription factor Xbp1, a mediator of the UPR [75]. Xbp1 is ubiquitously expressed at low levels, but its expression is dramatically upregulated in protein secreting cells such as ASCs. Xbp1 activity is further regulated by an unconventional splicing event that is also induced by the UPR [76]. In B cells, Pax5 is thought to repress Xbp1, with Pax5 down-regulation during ASC differentiation contributing to Xbp1 activation ([77], Fig. 3). Xbp1 was initially proposed to be essential for ASC development [78], however recent studies using a B cell specific knockout of Xbp1, have shown that ASCs form in its absence, but that they are strongly impaired in their ability to secrete high amounts of immunoglobulin [75,79]. Whether Xbp1 plays any role in the ASC differentiation pathway remains to be resolved, with one

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

345

Fig. 4. Graded expression of Irf4 during B cell differentiation. B cells from Blimp1-GFP reporter mice were stimulated with anti-CD40 and T cell cytokines (IL4 and IL5) for 4 days. (A) Shows a contour plot of B220 versus Blimp-GFP. In this plot B220hi cells (red box representing the gate, predominantly undivided cells) express low levels of Irf4, as shown by the red profile in (B). B220low and Blimp1-GFP− cells (blue box) have higher expression of Irf4, although this level is distributed over a broad range (blue). Blimp1-GFP+ ASCs (green box) elevate Irf4 even further (green profile).

study suggesting that Xbp1-deficient B cells expressed increased amounts of Irf4 and Blimp1 in vitro [79], while a second study found normal levels of these two factors in ASCs in vivo [75].

4. Towards describing a gene regulatory network controlling late B cell differentiation The large amount of data that was summarized in the preceding section was in the whole generated by the analysis of individual transcription factors in isolation. However, this information can also be used to construct gene regulatory networks that attempt to define the crucial processes and interactions that allow the transition from a B cell to an ASC, in a manner analogous to that successfully applied to early haematopoiesis and T cell differentiation [80,81]. B cell terminal differentiation is a particularly attractive system to study gene regulatory networks because of the clear gene expression boundary between B cells and ASCs and the documented antagonistic interactions between the major transcription factors involved. The first descriptions of the genetic network controlling B cell terminal differentiation were made by the Calame and Staudt laboratories [72,82–84]. These models were built on the finding that transcriptional repression reinforces mutually exclusive expression programs in B cells and ASCs (Fig. 3B). In this model, the B cell factors Bcl6, Pax5, and Mitf, directly repress the expression of the ASC regulators, Blimp1, Xbp1 and Irf4, respectively. Blimp1 also repressed the expression of Bcl6 and Pax5. The finding that Irf4 is critical for ASC differentiation through both repressing Bcl6 [35] and activating Blimp1 expression [53,55] has reinforced this model. More recently, the repressor Bach2 has been shown to function similarly, being activated in B cells by Pax5 [18] and then functioning primarily to repress premature Blimp1 expression [41]. While the genetic interactions mentioned above can be thought of as the primary events in the regulatory hierarchy of ASC gene expression, these factors are likely to directly regulate many cellular processes, including more specialized transcription factors that may sit lower in the regulatory hierarchy. For example, Pax5 directly activates the genes encoding a series of B cell factors, including Irf8, Spi-B, Aiolos and CIITA [17,18], many of which are involved in enabling the B cell response to antigen, cytokines, TLR ligands, or to undergo CSR and SHM [21], whereas Blimp1 represses a suite of factors including Id3, CIITA, Spi-B and Myc, whose silencing is associated with the terminal differentiation process [69,71,72]. It is also of note that several of these interactions, notably between Bcl6, Blimp1 and Irf4

have also been found to occur in other cell types including T cells [36,55,85–87]. While the mutual repression model nicely explains how the B cell and ASC program are sharply demarcated, it is less clear how the B cells overcome the repressive forces that block ASC differentiation. It is also challenging to integrate this deterministic model with evidence that the differentiation process is stochastic and divisionlinked. Finally, it is uncertain to what extent the process varies between the B cell subsets (B1, MZ and memory B cells) or as a result of distinct stimulation conditions in vitro and in vivo. We have attempted to address some of these questions by focussing on the regulation of Blimp1. Our analyses make use of a Blimp1gfp knock-in allele, where a GFP expression cassette is inserted into the Blimp1 locus [16]. Hematopoietic reconstitution with Blimp1gfp/gfp cells recapitulates the phenotype of mice with the B-cell-specific deletion of Blimp1 [68] in that they lack plasma cells and have severely reduced, but detectable, titres of all immunoglobulin isotypes [67]. In vitro and in vivo analysis of Blimp1gfp/gfp B cells revealed that in the absence of Blimp1 function, GFP+ B cells can be detected and that these cells show the characteristics of cells at an early stage of ASC differentiation, including reduced Pax5, activation of ASC-associated genes such as Igj, Xbp1, and low immunoglobulin secretion. These studies indicated that Blimp1-deficiency leads to a specific block at a stage of differentiation that we term the pre-plasmablast, and that Blimp1 is not required for the initiation of antibody secretion and the downregulation of Pax5, but is essential for the subsequent high-level immunoglobulin production that accompanies terminal ASC differentiation [67]. These findings raise an important question: What factor, if not Blimp1, initiates ASC differentiation? Analysis of B cells with normal Blimp1 function identified a phase prior to the onset of Blimp1-GFP expression in which Pax5 activity was inhibited. These data support a multistep model whereby ASC differentiation is initiated by the inhibition of Pax5 activity and ultimately transcription, followed by the induction of low-level immunoglobulin secretion and Blimp1 expression. Only subsequently does Blimp1 drive the full ASC terminal-differentiation program [67]. The identity of the initiating factor is as yet unknown, with Bcl6 [32], Obf1 [45] and Bach2 [40,41] unlikely to be candidates, as they are more directly involved in the regulation and timing of Blimp1 expression than in the maintenance of the B cell program through Pax5. Moreover Bcl6 is not required for ASC development in vivo or in vitro [37], despite being essential for GC formation, suggesting that Bcl6 plays a specific GC role that is overlaid on the core transcriptional network of

346

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

Fig. 5. A model to reconcile stochastic division-linked features of differentiation with deterministic gene regulatory networks. (A) Illustrates putative stochastic epigenetic regulation of the expression of two mutually antagonistic transcriptional regulators (depicted as red and green). A simulated FACS contour plot of the two regulators in B cells after activation is shown. Independent regulation and epigenetic effects ensure cells of every combination of high and low levels of each regulator are generated in the population. (B) Shows the fate of cells with different levels of the two regulators. In the first generation repression is dominant in each cell, however as the cells divide the ratios are altered allowing the repressor to become ineffective in some cells. The ratios and the modes of dilution lead to varying cell behaviour in different division rounds, including the possibility of never differentiating (top). The lightning motif indicates a differentiation event.

late B cell differentiation. In agreement with this conclusion, transgenic over-expression of Bcl6, while expanding GC size, does not block ASC development in vivo [88]. Irf4, on the other hand may potentially be the initiator of the process, as Irf4 expression is induced in activated B cells before Blimp1 and Irf4 has been shown to directly bind in both the Pax5 B-cell specific enhancer [52] and regulatory elements in the Blimp1 gene [53,55]. Irf4 is also proposed to repress Bcl6 in transformed GC B cell lines [35]. This process again may be GC specific, as multiple B cell lines and in vitro activated B cells co-express Bcl6 and Irf4. One potential scenario outlined below is that Irf4 regulates the ASC differentiation process in a concentration dependent manner [53]. 5. Blimp1 and Irf4 are dose-dependent regulators of antibody-secreting cell differentiation While Irf4 emerges as a key component in the decision to undergo CSR and develop into an ASC, it is necessary to contrast its action with the patterned division-linked changes seen at the population level. Questions to be addressed include: why do only a proportion of stimulated cells in vitro develop into ASCs and undergo CSR in each division, despite the presence of Irf4 in all activated B cells? If Irf4 plays a critical role for both CSR and ASC development, why are the two outcomes poorly correlated in the population [4,5]? How and why is the proportion of differentiated cells being changed with division? One potential approach to reconcile the cellular and molecular models of ASC differentiation may be to bring transcription factor concentration into the reckoning. In this regard, the clear changes in the levels of Irf4 that occur during the differentiation process may be informative. B cells express three distinct amounts of Irf4. Naïve resting B cells express a low basal amount of Irf4 that is rapidly increased upon antigenic or mitogenic stimulation in activated B cells, while ASC express a uniquely high level of Irf4 (Fig. 4, [53]). Ectopic Irf4 promotes ASC differentiation in a manner that correlates with concentration, suggesting that the relative abundance of Irf4 directly influences the differentiation potential of the activated B cells [53]. Furthermore, as shown in Fig. 4, the distribution of Irf4 in an activated B cell population is highly variable, following a typical log-Gaussian distribution that is consistent with stochastic epigenetic regulation within each cell. It is likely that this variegation of Irf4 levels in the population would render each cell more or less sensitive to the effects of the transcription factor, which could in turn explain the heterogeneity of cell fate even when cells are exposed to equivalent stimulatory signals. Interestingly, PU.1, a transcription factor that functions in a concentration

dependent manner in other hematopoietic lineages [89] and can cooperatively bind DNA with Irf4, also shows concentration dependence in its function. For example, removal of miRNA control of the Pu.1 3 untranslated region, or ectopic PU.1 expression inhibits ASC differentiation [8]. In addition to its absolute protein concentration, Irf4 may also be regulated by phosphorylation, providing yet another layer of complexity to the gene regulatory network [90]. How Irf4 levels are variably acquired with cell division becomes an important question. While concentration and stochastic variation in expression could contribute to the heterogeneity of cell fates, another mechanism that links cell division progression with the increased likelihood of differentiation must now be considered. A clue to the mechanism is suggested by recent work following single cell family trees. When stimulated with CpG, resting B cells undergo a varying number of division rounds (from two to five) before division senescence is reached and the cells die [91]. Hawkins et al. found that sibling progeny of a single founder cell divided the same number of times, strongly suggesting that the final division count was acquired and fixed prior to the first division and then passed to each descendant [92]. These data suggested a model where “division promoting factor(s)” accumulated in the first division were diluted evenly with division through each generation until they fell below a threshold required to promote cell division [93]. This study of cell division suggests how molecular regulators – such as promoters and dampeners of gene transcription – might accumulate in the first division and dilute with division. These changes together with stochastic variation in their acquired expression levels would mean that the proportions and ratios of factors would be heterogeneous in different cells and would further change with progressive division number (Fig. 5). A model built on this framework has the potential to integrate evidence for the stochastic elements of cell responses, the important role of cell division, and the deterministic gene regulatory network models for triggering cellular fates. In addition to Irf4 there is also evidence that the concentrations of Bach2 and Blimp1 impact on ASC differentiation [16,41]. In keeping with a direct and concentration-dependent role for Bach2 in the repression of Blimp1, LPS activated Bach2+/− B cells have increased Blimp1 and reduced CSR. Mathematical modelling of the Pax5–Bach2–Blimp1 interaction supports the notion that the concentration of Bach2 tunes the gene regulatory network, achieving a time-delay in Blimp1 induction and ASC differentiation [41]. Blimp1 expression is induced by a variety of stimuli that promote B cell differentiation, including LPS and cytokines [16,94]. Analysis of the Blimp1-GFP reporter mice revealed that in vivo, ASCs could be divided into two fractions, characterized by

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

intermediate (Blimp1int ) and high (Blimp1hi ) Blimp1 expression [16]. While both fractions are found in the spleen, Blimp1hi ASCs dominate in the bone marrow. These distinct Blimp1 levels are associated with differential expression of receptor molecules and functional differences, such as turnover and migratory capacities. They indicate that Blimp1int ASCs are short-lived and rapidly proliferating plasmablasts while the Blimp1hi population consists of long-lived plasma cells [16]. The relationship between these subsets is not fully understood. While the majority of plasmablasts die in an early phase of an immune reaction, it is likely that some cells proceed into the Blimp1hi long-lived ASC compartment in the spleen and bone marrow. Interestingly, it appears that ASCs localize to the bone marrow before up-regulating Blimp1, as migratory ASCs in the blood and spleen are Blimp1int [95,96]. These studies suggest that the concentration of Blimp1 plays a role in the terminal differentiation of cycling plasmablasts into quiescent and long-lived plasma cells. The signals responsible for these distinct Blimp1 levels, and the molecular targets of Blimp1 that mediate these functions remain important gaps in our understanding of the ASC differentiation process. The examples of Irf4 and Blimp1 demonstrate that the gene regulatory network that controls ASC differentiation is not static and that the relative concentrations and potential functionality of the regulators must be taken into account. Much remains to be done in this area, as reagents that allow accurate measurement of transcription factors such as monoclonal antibodies and fluorescently tagged proteins are currently available for only a minority of these proteins. Moreover, it is mostly unknown how changes in transcription factor abundance results in altered target gene selection. Genome wide analysis of the occupied DNA binding sites for a range of transcription factor concentrations will be required to fully begin to incorporate this variable into our understanding of gene regulatory networks.

6. Conclusions and future perspectives Research over the past decade has provided a wealth of data to highlight the key factors in the gene regulatory network driving the terminal differentiation of B cells. Two classes of transcription factors are required, those that promote the B cell state, such as Bcl6 and Pax5 and those including Irf4 and Blimp1, that favour ASC differentiation. On a genetic level, the mutual antagonism between these factors ensures that these key developmental stages in B cell differentiation are kept transcriptionally and functionally distinct. This model is an over-simplification, as many processes are not absolute. Relative protein concentrations and protein–protein interactions play a role that has not been entirely appreciated. It is also clear that terminal differentiation is neither a predetermined outcome, nor an inevitable consequence of activation. Rather it is carefully regulated and tethered to the process of clonal expansion, guaranteed to apportion cells broadly within the range of possible fates. The challenge now is to integrate the molecular and cellular approaches to guide development of quantitative models of the gene regulatory networks. This integration will, in the future, more accurately describe the production of a diverse array of isotypeswitched activated B cells, memory B cells and antibody-secreting plasmablasts and plasma cells that occurs during a natural antibody response. The ability to map the DNA binding sites occupied in vivo by a given transcription factor throughout the genome, as well as to accurately profile factor dependent gene expression changes provides one way forward for the field. The application of gene regulatory network analysis also requires further investment in tools to visualize transcription factor concentration on a single cell level and mathematical approaches to take into account the probabilistic nature of biological phenomena. Finally, video microscopic

347

tracking approaches may be required to discern the differentiation processes on a single cell and clonal level. Acknowledgements We would like to thank members of the Walter and Eliza Hall Institute B cell program for discussions. This research was supported by the Pfizer Australia Research Fellowship and ARC Future Fellowship to S.L.N. and National Health and Medical Research Council of Australia Research Fellowships to P.D.H. and L.M.C. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIIS. References [1] Fairfax KA, Kallies A, Nutt SL, Tarlinton DM. Plasma cell development: from B-cell subsets to long-term survival niches. Semin Immunol 2008;20:49–58. [2] Fairfax KA, Corcoran LM, Pridans C, Huntington ND, Kallies A, Nutt SL, et al. Different kinetics of blimp-1 induction in B cell subsets revealed by reporter gene. J Immunol 2007;178:4104–11. [3] Hodgkin PD, Lee JH, Lyons AB. B cell differentiation and isotype switching is related to division cycle number. J Exp Med 1996;184:277–81. [4] Deenick EK, Hasbold J, Hodgkin PD. Switching to IgG3, IgG2b, and IgA is division linked and independent, revealing a stochastic framework for describing differentiation. J Immunol 1999;163:4707–14. [5] Hasbold J, Corcoran LM, Tarlinton DM, Tangye SG, Hodgkin PD. Evidence from the generation of immunoglobulin G-secreting cells that stochastic mechanisms regulate lymphocyte differentiation. Nat Immunol 2004;5:55–63. [6] Calame KL, Lin KI, Tunyaplin C. Regulatory mechanisms that determine the development and function of plasma cells. Annu Rev Immunol 2003;21:205–30. [7] Kallies A, Nutt SL. Terminal differentiation of lymphocytes depends on Blimp-1. Curr Opin Immunol 2007;19:156–62. [8] Vigorito E, Perks KL, Abreu-Goodger C, Bunting S, Xiang Z, Kohlhaas S, et al. microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells. Immunity 2007;27:847–59. [9] Lee CH, Melchers M, Wang H, Torrey TA, Slota R, Qi CF, et al. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein. J Exp Med 2006;203:63–72. [10] Lin L, Gerth AJ, Peng SL. Active inhibition of plasma cell development in resting B cells by microphthalmia-associated transcription factor. J Exp Med 2004;200:115–22. [11] John SA, Clements JL, Russell LM, Garrett-Sinha LA. Ets-1 regulates plasma cell differentiation by interfering with the activity of the transcription factor Blimp1. J Biol Chem 2008;283:951–62. [12] Cobaleda C, Schebesta A, Delogu A, Busslinger M. Pax5: the guardian of B cell identity and function. Nat Immunol 2007;8:463–70. [13] Fuxa M, Busslinger M. Reporter gene insertions reveal a strictly B lymphoidspecific expression pattern of pax5 in support of its B cell identity function. J Immunol 2007;178:3031–7. [14] Nutt SL, Heavey B, Rolink AG, Busslinger M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 1999;401:556–62. [15] Cobaleda C, Jochum W, Busslinger M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors. Nature 2007;449:473–7. [16] Kallies A, Hasbold J, Tarlinton DM, Dietrich W, Corcoran LM, Hodgkin PD, et al. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J Exp Med 2004;200:967–77. [17] Pridans C, Holmes ML, Polli M, Wettenhall JM, Dakic A, Corcoran LM, et al. Identification of Pax5 target genes in early B cell differentiation. J Immunol 2008;180:1719–28. [18] Schebesta A, McManus S, Salvagiotto G, Delogu A, Busslinger GA, Busslinger M. Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function. Immunity 2007;27:49–63. [19] Delogu A, Schebesta A, Sun Q, Aschenbrenner K, Perlot T, Busslinger M. Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells. Immunity 2006;24:269–81. [20] Holmes ML, Carotta S, Corcoran LM, Nutt SL. Repression of Flt3 by Pax5 is crucial for B-cell lineage commitment. Genes Dev 2006;20:933–8. [21] Holmes ML, Pridans C, Nutt SL. The regulation of the B-cell gene expression programme by Pax5. Immunol Cell Biol 2008;86:47–53. [22] Nera KP, Kohonen P, Narvi E, Peippo A, Mustonen L, Terho P, et al. Loss of Pax5 promotes plasma cell differentiation. Immunity 2006;24:283–93. [23] Basso K, Dalla-Favera R. BCL6: master regulator of the germinal center reaction and key oncogene in B cell lymphomagenesis. Adv Immunol 2010;105:193–210. [24] Duy C, Yu JJ, Nahar R, Swaminathan S, Kweon SM, Polo JM, et al. BCL6 is critical for the development of a diverse primary B cell repertoire. J Exp Med 2010;207:1209–21.

348

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

[25] Cattoretti G, Chang CC, Cechova K, Zhang J, Ye BH, Falini B, et al. BCL-6 protein is expressed in germinal-center B cells. Blood 1995;86:45–53. [26] Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM. Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science 1997;276:589–92. [27] Ye BH, Cattoretti G, Shen Q, Zhang J, Hawe N, de Waard R, et al. The BCL-6 protooncogene controls germinal-centre formation and Th2-type inflammation. Nat Genet 1997;16:161–70. [28] Fukuda T, Yoshida T, Okada S, Hatano M, Miki T, Ishibashi K, et al. Disruption of the Bcl6 gene results in an impaired germinal center formation. J Exp Med 1997;186:439–48. [29] Cattoretti G, Pasqualucci L, Ballon G, Tam W, Nandula SV, Shen Q, et al. Deregulated BCL6 expression recapitulates the pathogenesis of human diffuse large B cell lymphomas in mice. Cancer Cell 2005;7:445–55. [30] Basso K, Saito M, Sumazin P, Margolin AA, Wang K, Lim WK, et al. Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal center B cells. Blood 2010;115:975–84. [31] Ci W, Polo JM, Cerchietti L, Shaknovich R, Wang L, Yang SN, et al. The BCL6 transcriptional program features repression of multiple oncogenes in primary B cells and is deregulated in DLBCL. Blood 2009;113:5536–48. [32] Tunyaplin C, Shaffer AL, Angelin-Duclos CD, Yu X, Staudt LM, Calame KL. Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation. J Immunol 2004;173:1158–65. [33] Linterman MA, Beaton L, Yu D, Ramiscal RR, Srivastava M, Hogan JJ, et al. IL21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J Exp Med 2010;207:353–63. [34] Zotos D, Coquet JM, Zhang Y, Light A, D’Costa K, Kallies A, et al. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. J Exp Med 2010;207:365–78. [35] Saito M, Gao J, Basso K, Kitagawa Y, Smith PM, Bhagat G, et al. A signaling pathway mediating downregulation of BCL6 in germinal center B cells is blocked by BCL6 gene alterations in B cell lymphoma. Cancer Cell 2007;12: 280–92. [36] Cimmino L, Martins GA, Liao J, Magnusdottir E, Grunig G, Perez RK, et al. Blimp1 attenuates Th1 differentiation by repression of ifng, tbx21, and bcl6 gene expression. J Immunol 2008;181:2338–47. [37] Toyama H, Okada S, Hatano M, Takahashi Y, Takeda N, Ichii H, et al. Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 2002;17:329–39. [38] Muto A, Hoshino H, Madisen L, Yanai N, Obinata M, Karasuyama H, et al. Identification of Bach2 as a B-cell-specific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3 enhancer. EMBO J 1998;17:5734–43. [39] Muto A, Tashiro S, Nakajima O, Hoshino H, Takahashi S, Sakoda E, et al. The transcriptional programme of antibody class switching involves the repressor Bach2. Nature 2004;429:566–71. [40] Ochiai K, Muto A, Tanaka H, Takahashi S, Igarashi K. Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6. Int Immunol 2008;20:453–60. [41] Muto A, Ochiai K, Kimura Y, Itoh-Nakadai A, Calame KL, Ikebe D, et al. Bach2 represses plasma cell gene regulatory network in B cells to promote antibody class switch. EMBO J 2010;29:4048–61. [42] Humbert PO, Corcoran LM. oct-2 gene disruption eliminates the peritoneal B-1 lymphocyte lineage and attenuates B-2 cell maturation and function. J Immunol 1997;159:5273–84. [43] Corcoran LM, Karvelas M. Oct-2 is required early in T cell-independent B cell activation for G1 progression and for proliferation. Immunity 1994;1: 635–45. [44] Schubart K, Massa S, Schubart D, Corcoran LM, Rolink AG, Matthias P. B cell development and immunoglobulin gene transcription in the absence of Oct-2 and OBF-1. Nat Immunol 2001;2:69–74. [45] Corcoran LM, Hasbold J, Dietrich W, Hawkins E, Kallies A, Nutt SL, et al. Differential requirement for OBF-1 during antibody-secreting cell differentiation. J Exp Med 2005;201:1385–96. [46] Emslie D, D’Costa K, Hasbold J, Metcalf D, Takatsu K, Hodgkin PO, et al. Oct2 enhances antibody-secreting cell differentiation through regulation of IL-5 receptor alpha chain expression on activated B cells. J Exp Med 2008;205:409–21. [47] Shaffer AL, Emre NC, Romesser PB, Staudt LM. IRF4: immunity. Malignancy! Therapy? Clin Cancer Res 2009;15:2954–61. [48] Mittrucker HW, Matsuyama T, Grossman A, Kundig TM, Potter J, Shahinian A, et al. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 1997;275:540–3. [49] Brass AL, Zhu AQ, Singh H. Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers. EMBO J 1999;18:977–91. [50] Brass AL, Kehrli E, Eisenbeis CF, Storb U, Singh H. Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1. Genes Dev 1996;10:2335–47. [51] Pongubala JM, Nagulapalli S, Klemsz MJ, McKercher SR, Maki RA, Atchison ML. PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3 enhancer activity. Mol Cell Biol 1992;12:368–78. [52] Decker T, Pasca di Magliano M, McManus S, Sun Q, Bonifer C, Tagoh H, et al. Stepwise activation of enhancer and promoter regions of the B cell commitment gene Pax5 in early lymphopoiesis. Immunity 2009;30:508–20.

[53] Sciammas R, Shaffer AL, Schatz JH, Zhao H, Staudt LM, Singh H. Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation. Immunity 2006;25:225–36. [54] Klein U, Casola S, Cattoretti G, Shen Q, Lia M, Mo T, et al. Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination. Nat Immunol 2006;7:773–82. [55] Kwon H, Thierry-Mieg D, Thierry-Mieg J, Kim H-P, Oh J, Tunyaplin C, et al. Analysis of IL-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors. Immunity 2009;31:941–52. [56] Lu R, Medina KL, Lancki DW, Singh H. IRF-4,8 orchestrate the pre-B-to-B transition in lymphocyte development. Genes Dev 2003;17:1703–8. [57] Feng J, Wang H, Shin DM, Masiuk M, Qi CF, Morse III HC. IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools. J Immunol 2011;186:1458–66. [58] Takaoka A, Yanai H, Kondo S, Duncan G, Negishi H, Mizutani T, et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 2005;434:243–9. [59] Paun A, Reinert JT, Jiang Z, Medin C, Balkhi MY, Fitzgerald KA, et al. Functional characterization of murine interferon regulatory factor 5 (IRF-5) and its role in the innate antiviral response. J Biol Chem 2008;283:14295–308. [60] Lien C, Fang CM, Huso D, Livak F, Lu R, Pitha PM. Critical role of IRF-5 in regulation of B-cell differentiation. Proc Natl Acad Sci USA 2010;107:4664–8. [61] Nutt SL, Fairfax KA, Kallies A. BLIMP1 guides the fate of effector B and T cells. Nat Rev Immunol 2007;7:923–7. [62] Chan YH, Chiang MF, Tsai YC, Su ST, Chen MH, Hou MS, et al. Absence of the transcriptional repressor Blimp-1 in hematopoietic lineages reveals its role in dendritic cell homeostatic development and function. J Immunol 2009;183:7039–46. [63] Chang DH, Angelin-Duclos C, Calame K. BLIMP-1: trigger for differentiation of myeloid lineage. Nat Immunol 2000;1:169–76. [64] Kallies A, Carotta S, Huntington ND, Bernard NJ, Tarlinton DM, Smyth MJ, et al. A role for Blimp-1 in the transcriptional network controlling natural killer cell maturation. Blood 2010;117:869–79. [65] Smith MA, Maurin M, Cho HI, Becknell B, Freud AG, Yu J, et al. PRDM1/Blimp1 controls effector cytokine production in human NK cells. J Immunol 2010;185:6058–67. [66] Gonzalez-Garcia I, Ocana E, Jimenez-Gomez G, Campos-Caro A, Brieva JA. Immunization-induced perturbation of human blood plasma cell pool: progressive maturation IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigen-specific and nonspecific plasma cells. J Immunol 2006;176:4042–50. [67] Kallies A, Hasbold J, Fairfax K, Pridans C, Emslie D, McKenzie BS, et al. Initiation of plasma-cell differentiation is independent of the transcription factor Blimp1. Immunity 2007;26:555–66. [68] Shapiro-Shelef M, Lin KI, McHeyzer-Williams LJ, Liao J, McHeyzer-Williams MG, Calame K. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 2003;19:607–20. [69] Piskurich JF, Lin KI, Lin Y, Wang Y, Ting JP, Calame K. BLIMP-I mediates extinction of major histocompatibility class II transactivator expression in plasma cells. Nat Immunol 2000;1:526–32. [70] Lin KI, Angelin-Duclos C, Kuo TC, Calame K. Blimp-1-dependent repression of Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma cells. Mol Cell Biol 2002;22:4771–80. [71] Lin KI, Lin Y, Calame K. Repression of c-myc is necessary but not sufficient for terminal differentiation of B lymphocytes in vitro. Mol Cell Biol 2000;20:8684–95. [72] Shaffer AL, Lin KI, Kuo TC, Yu X, Hurt EM, Rosenwald A, et al. Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program. Immunity 2002;17:51–62. [73] Doody GM, Care MA, Burgoyne NJ, Bradford JR, Bota M, Bonifer C, et al. An extended set of PRDM1/BLIMP1 target genes links binding motif type to dynamic repression. Nucleic Acids Res 2010;38:5336–50. [74] Kuo TC, Calame KL. B lymphocyte-induced maturation protein (Blimp)-1 IFN regulatory factor (IRF)-1, and IRF-2 can bind to the same regulatory sites. J Immunol 2004;173:5556–63. [75] Todd DJ, Lee AH, Glimcher LH. The endoplasmic reticulum stress response in immunity and autoimmunity. Nat Rev Immunol 2008;8:663–74. [76] Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 2001;107:881–91. [77] Reimold AM, Ponath PD, Li YS, Hardy RR, David CS, Strominger JL, et al. Transcription factor B cell lineage-specific activator protein regulates the gene for human X-box binding protein 1. J Exp Med 1996;183:393–401. [78] Reimold AM, Iwakoshi NN, Manis J, Vallabhajosyula P, Szomolanyi-Tsuda E, Gravallese EM, et al. Plasma cell differentiation requires the transcription factor XBP-1. Nature 2001;412:300–7. [79] Hu CC, Dougan SK, McGehee AM, Love JC, Ploegh HL. XBP-1 regulates signal transduction, transcription factors and bone marrow colonization in B cells. EMBO J 2009;28:1624–36. [80] Laslo P, Pongubala JM, Lancki DW, Singh H. Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system. Semin Immunol 2008;20:228–35. [81] Rothenberg EV, Zhang J, Li L. Multilayered specification of the T-cell lineage fate. Immunol Rev 2010;238:150–68. [82] Shaffer AL, Shapiro-Shelef M, Iwakoshi NN, Lee AH, Qian SB, Zhao H, et al. XBP1, downstream of Blimp-1, expands the secretory apparatus and other

S.L. Nutt et al. / Seminars in Immunology 23 (2011) 341–349

[83]

[84] [85]

[86] [87]

[88]

[89]

organelles, and increases protein synthesis in plasma cell differentiation. Immunity 2004;21:81–93. Shaffer AL, Yu X, He Y, Boldrick J, Chan EP, Staudt LM. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity 2000;13:199–212. Shapiro-Shelef M, Calame K. Regulation of plasma-cell development. Nat Rev Immunol 2005;5:230–42. Cretney E, Xin A, Shi W, Minnich M, Masson F, Miasari M, et al. The transcription factors Blimp1 and IRF4 jointly control differentiation and function of effector regulatory T cells. Nat Immunol 2011;12:304–11. Crotty S, Johnston RJ, Schoenberger SP. Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation. Nat Immunol 2010;11:114–20. Miyauchi Y, Ninomiya K, Miyamoto H, Sakamoto A, Iwasaki R, Hoshi H, et al. The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis. J Exp Med 2010;207:751–62. Cattoretti G, Angelin-Duclos C, Shaknovich R, Zhou H, Wang D, Alobeid B. PRDM1/Blimp-1 is expressed in human B-lymphocytes committed to the plasma cell lineage. J Pathol 2005;206:76–86. Carotta S, Wu L, Nutt SL. Surprising new roles for PU.1 in the adaptive immune response. Immunol Rev 2010;238:63–75.

349

[90] Biswas PS, Gupta S, Chang E, Song L, Stirzaker RA, Liao JK, et al. Phosphorylation of IRF4 by ROCK2 regulates IL-17 and IL-21 production and the development of autoimmunity in mice. J Clin Invest 2010;120:3280–95. [91] Turner ML, Hawkins ED, Hodgkin PD. Quantitative regulation of B cell division destiny by signal strength. J Immunol 2008;181:374–82. [92] Hawkins ED, Markham JF, McGuinness LP, Hodgkin PD. A single-cell pedigree analysis of alternative stochastic lymphocyte fates. Proc Natl Acad Sci USA 2009;106:13457–62. [93] Markham JF, Wellard CJ, Hawkins ED, Duffy KR, Hodgkin PD. A minimum of two distinct heritable factors are required to explain correlation structures in proliferating lymphocytes. J R Soc Interface 2010;7:1049–59. [94] Sciammas R, Davis MM. Blimp-1; immunoglobulin secretion and the switch to plasma cells. Curr Top Microbiol Immunol 2005;290:201–24. [95] Blink EJ, Light A, Kallies A, Nutt SL, Hodgkin PD, Tarlinton DM. Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization. J Exp Med 2005;201:545–54. [96] Kabashima K, Haynes NM, Xu Y, Nutt SL, Allende ML, Proia RL, et al. Plasma cell S1P1 expression determines secondary lymphoid organ retention versus bone marrow tropism. J Exp Med 2006;203:2683–90.