REVIEW doi: 10.1111/j.1365-3083.2006.01809.x ..................................................................................................................................................................
Pax5 – a Critical Inhibitor of Plasma Cell Fate K.-P. Nera & O. Lassila
Abstract Turku Graduate School of Biomedical Sciences, Department of Medical Microbiology, University of Turku, Turku, Finland Received 9 May 2006; Accepted in revised form 6 June 2006 Correspondence to: Dr K.-P. Nera, Turku Graduate School of Biomedical Sciences, University of Turku, Kiinamyllynkatu 13, 20500 Turku, Finland. E-mail:
[email protected]
Paired box protein 5 (Pax5) is essential for early B cell commitment as well as for B cell development, and continuous expression of Pax5 is required throughout the B cell lineage to maintain the functional identity of B cells. During B cell activation, Pax5 is downregulated before terminal differentiation into antibody-secreting plasma cells, and enforced expression of Pax5 prevents plasmacytic development. Recently, loss of Pax5 was shown to result in the substantial transition to a plasma cell state, demonstrating a functionally significant role for Pax5 in the regulation of terminal B cell differentiation. Here we elucidate the current understanding about the function of Pax5 as a key inhibitor of plasma cell differentiation.
Introduction In the humoral immune response, antigen-driven B cell activation leads to terminal differentiation producing plasma cells, the final effector cells of B lymphoid lineage. Plasma cells produce large quantities of soluble immunoglobulin (Ig), which execute a crucial role in the adaptive immune system. During plasma cell differentiation, most B cell characteristics are lost while coordinated changes eventually lead to a cellular structure of non-dividing highly secreting cells. These radical changes in the phenotype are ultimately regulated by an interactive network of transcription factors, which appear to have an intrinsic function to either promote or inhibit plasma cell differentiation. The key regulators promoting the fate of plasma cells are two transcription factors: B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box binding protein 1 (XBP-1) [1–3]. Both these factors are essential for plasma cell differentiation [4, 5]. Blimp-1 is considered the master plasma cell factor, as it shuts down the B cell gene expression programme [6] and is alone sufficient to induce the development of plasma cells [4, 7]. In contrast, XBP-1 is not self-sufficient to induce plasma cell fate in the absence of Blimp-1 [4], but appears to be essential for the expanded secretory apparatus, increased protein synthesis and secretion in plasma cells [8]. The transcription factors Pax5 (also known as B-cell-specific activation protein, BSAP) and B-cell lymphoma 6 protein (Bcl-6) are the main inhibitors of plasma cell differentiation, as both prevent B cell activation and plasma cell formation [9–12]. Pax5 has been shown to act as the
factor maintaining the B cell identity [13, 14], while also inhibiting the expression of XBP-1 [15], whereas Bcl-6 is thought to directly suppress Blimp-1 induction [16, 17]. Given that Blimp-1 appears to contribute to the downregulation of Bcl-6 [6] and directly represses Pax5 expression [12], Bcl-6 has been regarded as the central inhibitor of plasma cell fate, hence supporting a model in which downregulation or degradation of Bcl-6 is the primary event in the induction of plasmacytic differentiation. However, a loss of Pax5 was recently shown to promote plasma cell differentiation, as inactivation of Pax5 from a B cell line resulted in the substantial loss of B cell identity with concomitant induction of Ig secretion and plasma cell gene expression programme, i.e. downregulation of Bcl-6 with a simultaneous increase in the expression of Blimp-1 and XBP-1 [18]. Moreover, the depletion of Pax5 from the mature mouse B cells led to the induction of Blimp-1, although neither downregulation of Bcl-6 nor upregulation of XBP-1 was observed [19]. Nevertheless, these recent findings provide new insight into the significance of Pax5 in the terminal differentiation of B cells [20]. We discuss how the role of Pax5 in the regulation of B cell fate has evolved from the B cell commitment and maintenance factor to a factor with much broader functional importance in the late events of B cell differentiation. We also address how this new role of Pax5 as one of the key inhibitors of plasma cell differentiation provides potential plasticity to the complex regulatory network controlling B cell activation and plasma cell differentiation. 2006 The Authors
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Early B cell commitment and maintenance of B cell characteristics are dependent on Pax5 Our knowledge concerning the functional importance of transcription factors guiding B cell lineage is, to a great extent, based on the mouse knockout studies, which have defined several developmental blocks due to an absence of a single transcriptional regulator [21, 22]. Pax5 seems to function downstream of E2A and early B cell factor (EBF) [23, 24]. Although, the B cell-specific gene expression programme appears to be initiated by E2A and EBF, the commitment to the B cell lineage is not ensured unless Pax5 is expressed. In the bone marrow of Pax5deficient mice, B cell development is arrested at an early pro-B cell stage of the development [25], and Pax5)/) B cell progenitors exhibit characteristics of uncommitted cells by still retaining a broad developmental potential [26–28]. In appropriate in vitro culture conditions, Pax5)/) pro-B cells are able to differentiate into functional macrophages, dendritic cells, natural killer cells, granulocytes and osteoclasts [26]. In addition to these cell types, Pax5)/) pro-B cells injected into sublethally irradiated mice give rise to functional T cells, neutrophils, and erythrocytes in vivo [26–28], but the multilineage potential is suppressed by restoration of Pax5 expression, which also rescues the pro-B cell arrest of Pax5)/) mice [26]. Hence, Pax5 has been considered a critical commitment factor of B cell lineage that ultimately restricts the precursor cells to the B cell fate. The fact that Pax5 remains to be expressed throughout the B cell lineage up until the activation and terminal differentiation of plasma cells [29, 30] suggests that Pax5 has an important role also after early B cell commitment. In accordance, conditional gene targeting studies in mice have revealed that continuous Pax5 expression is critical for the maintenance of B cell population [13, 14], as conditional inactivation of Pax5 leads to the total loss of identity and function of mature mouse B cells [13], whereas the conditionally Pax5-deficient pro-B cells show indications for reversion of the B cell phenotype [14]. The role of Pax5 during B cell development appears to be dualistic, as Pax5 promotes the activity of many B cell-specific genes and simultaneously participates in the repression of genes, which are inappropriate for the B cell lineage [21]. Pax5 can act either as an activator or as a repressor, which is affected by interactions with distinct partner proteins that determine the transcriptional activity of Pax5. Interactions with co-repressors convert Pax5 from a transcriptional activator to a repressor [31, 32]. During early B cell development Pax5 prevents myeloid and T-lymphoid differentiation by mediating the repression of M-CSFR and Notch1 [26, 33]. However, ectopic Pax5 expression in myeloid lineage is not sufficient to prevent early myelopoiesis [33, 34], and the mechanism
through which Pax5 participates in the suppression of B lineage inappropriate genes remains obscure, although some evidence exists that Pax5 interferes with the action of myeloid-specific transcription factors during B cell lineage restriction [35]. Pax5 facilitates the commitment of B cell progenitors by activating the transcription of Iga [36, 37], BLNK [38] and CD19 [37, 39], all of which function in the preBCR signalling pathway constituting a critical developmental checkpoint in B cell differentiation. Moreover, Pax5 affects Ig recombination, as it controls the second VH-DJH recombination step of the IgH gene [40, 41], Pax5/c-Myb/LEF-1 complex cooperatively binds and activates the RAG2 promoter [42–44], and evidence exists that Pax5 is essential for sterile j transcription during Igj chain gene rearrangement [45, 46]. Furthermore, distal VH-DJH recombination and locus contraction of IgH can also be induced in T cells by ectopic Pax5 expression [47, 48]. Hence, Pax5 regulates multiple aspects of functional B cell identity by promoting the expression of BCR signalling machinery and Ig recombination, while at the same time repressing the lineage inappropriate gene expression programmes.
The elimination of Pax5 is essential for plasma cell differentiation Pax5 is needed to maintain the identity of B cells [13, 14] and thus remains to be expressed throughout the B cell lineage [29, 30]. However, following the antigen-driven B cell activation, Blimp-1 is thought to directly downregulate Pax5 expression [12]. The Blimp-1-mediated repression of Pax5 appears to have a critical importance in plasmacytic differentiation, as the enforced expression of Pax5 prevents the development of functional plasma cells [9, 10, 12]. Pax5 has also been shown to repress the expression of IgH [49], J chain [50–52] and XBP-1 [15], which are all genes associated with Ig secretion [1–3]. This provides one potential explanation for the importance to downregulate Pax5 expression during plasmacytic differentiation. XBP-1 is required for the development of functional plasma cells and Ig secretion [5, 53], as it is essential for the induction of multiple genes, which are indispensable for the effective protein secretion [8]. Similar to ectopic Blimp-1 expression [54], enforced XBP-1 expression in a B cell line can initiate plasma cell differentiation [5]. Whereas overexpression of XBP-1 in B cell lines expands the secretory apparatus [8], XBP-1-deficient B cells cannot become Ig-secreting plasma cells [5]. Plasma cell differentiation includes a great increase in the amount of Ig heavy and light chain proteins in the endoplasmic reticulum (ER). Unfolded protein response (UPR) allows cells to adjust to the stress caused by a large protein content of ER [55]. The initial expansion of ER in the UPR
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occurs prior to XBP-1 expression [56], and ER stress leads to the induction of XBP-1 by activating transcription factor 6 (ATF-6) [57], which is induced in response to endoplasmic stress. Moreover, XBP-1 mRNA is spliced by IRE1 in response to ER stress, and only the spliced form of XBP-1 can contribute to Ig secretion and UPR [53, 58], where XBP-1 regulates important ER stress-related chaperones [59]. While ATF-6 as well as IL-6 signalling are both considered to promote XBP-1 induction [57, 60], Pax5 appears to serve as a factor suppressing the premature induction of XBP-1 [15]. Given the critical role of XBP-1 in protein secretion [5, 8, 53] and the fact that Pax5 suppresses both XBP-1 expression [15] and plasma cell differentiation [9, 10, 12], it is evident that Pax5 expression has to be eliminated for the successful development of functional plasma cells. XBP-1 is thought to function downstream of Blimp-1 [4, 8], as restored XBP-1 expression cannot promote plasmacytic differentiation in the absence of Blimp1 [4], and while Blimp-1 is expressed in XBP-1)/) B cells, Blimp-1-deficient B cells fail to express XBP-1 [4, 8]. On the other hand, enforced expression of Blimp-1 in B cell lines leads to an induction of XBP-1 and downregulation of Pax5 [6], a direct target for Blimp-1-mediated repression [12]. This may indicate that Blimp-1 inhibits the expression of Pax5 hence promoting the induction of XBP-1. Although Blimp-1-mediated downregulation of Pax5 is considered to be necessary for increased XBP-1 expression [1–3], it is evidently not enough for a full induction of XBP-1, as ectopic Blimp-1 expression and consequent downregulation of Pax5 in a B cell line induced only a moderate increase in XBP-1 expression, which was not comparable to a level of XBP-1 in plasma cells [6]. Therefore, full induction and activation of XBP1 appears to be influenced by both: (i) removal of repression activity of Pax5 by Blimp-1 [12, 15], as well as (ii) events promoting the activity of XBP-1, i.e. ATF-6 induction [57], IL-6 signalling [60] and XBP-1 splicing by IRE1 [58]. All mature B cell subsets – B1 cells, marginal zone B cells and follicular (B2) B cells – are able to give rise to plasma cells upon activation [1–3]. Activated B cells undergo a strong proliferative burst, and usually a robust proliferation of plasmablasts precedes the terminal differentiation into plasma cells [61]. After the initial proliferation, both marginal zone and follicular B cells can directly develop to plasmablasts and eventually produce plasma cells [3], but these plasma cells do not somatically hypermutate their Ig genes [62]. Alternatively, activated follicular B cells may also establish germinal centres (GC), which are specialized areas in the follicle where somatic hypermutation (SHM) and class-switch recombination (CSR) occur. In GCs, activated B cells undergo cycles of proliferation [63] accompanied by affinity maturation via SHM [63, 64] and selection events through
apoptosis [65]. As a result, plasmablasts producing antibodies with increased affinity for antigen and switched isotypes exit GCs to differentiate into plasma cells. During the GC reaction, premature activation of Blimp-1 is suppressed by Bcl-6 protein [11, 16, 17]. In contrast to Pax5-deficient mice [25], B cells develop normally in the mice deficient for Bcl-6 [66, 67], but Bcl-6 is needed for GC formation [66–68] and activated B cells fail to undergo GC reaction in the absence of Bcl-6 [66, 67]. Despite the fact that both resting B cells and GC B cells have comparable Bcl-6 expression at the mRNA level [69], GC B cells exhibit increased amounts of Bcl-6 protein compared with the resting B cells [69, 70]. This is also in accordance with the impaired GC reaction of Bcl-6-deficient mice [66, 67]. Apparently, the amount of Bcl-6 in B cells is regulated via complex mechanisms that occur at multiple regulatory levels. Upon B cell activation, Bcl-6 is transcriptionally downregulated, although not in GC B cells [69], where the level of Bcl-6 protein is clearly the highest [69, 70]. Moreover, the evidence that the gene encoding Bcl-6 is potentially subjected for negative autoregulation by its own products [71] adds further complexity to the transcriptional regulation of Bcl-6. On the other hand, Bcl-6 expression is also regulated post-transcriptionally, as B cell receptor (BCR) signalling leads to mitogen-activated protein kinasemediated phosphorylation and degradation of Bcl-6 protein [72], thus contributing to the subsequent differentiation towards the terminal plasma cell fate. Prior to terminal differentiation, Bcl-6 is thought to promote the essential expansion and self-renewing potential of GC B cells [73–75], where the evident demand for continuous Bcl-6 expression has been established by gene targeting studies in mice [66–68]. Furthermore, cytokines contribute to the self-renewal capacity of GC B cells as well as memory B cells via signal transducer of activation and transcription 5 (STAT5)-mediated Bcl-6 induction [76]. Nevertheless, the complex regulation of Bcl-6 in B cells is not yet understood. Moreover, despite the fact that both Pax5 and Bcl-6 are expressed in GCs (Fig. 1), and either of them alone appears to be sufficient to prevent plasmacytic differentiation according to ectopic expression studies [9–12], only limited information about the relationship between these two major factors inhibiting the plasma cell fate has been obtained.
Inactivation of Pax5 induces a spontaneous plasmacytic differentiation Recently, we disrupted both Pax5-alleles from the DT40 B cell line [18] to gain further insight into the functional importance of Pax5 during the late events of B cell lineage differentiation. The chicken B cell lymphoma line DT40 is surface IgM positive and appears to be arrested at the bursal B cell stage [77, 78], as the gene expression 2006 The Authors
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Figure 1 Expression of major transcriptional regulators of plasma cell development during the different stages of B cell activation. After B cell activation, the activated B cells proliferate and either develop directly to proliferating plasmablasts or alternatively form germinal centres (GC), where proliferating GC B cells undergo affinity maturation involving activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) and class switch recombination (CSR). B cells with high affinity for antigen and switched Ig isotypes exit GCs and differentiate via proliferating plasmablasts into highly secreting plasma cells. Pax5 and Bcl-6 inhibit plasma cell differentiation, whereas Blimp-1 and XBP-1 are the key regulators promoting plasmacytic development. The horizontal blue bars show the expression of these transcription factors during the different stages of terminal B cell differentiation. *Although Bcl-6 is expressed at the mRNA level also in resting B cells, high levels of Bcl-6 protein are found only from GC B cells. §XBP-1 expression represents the spliced active form of XBP-1.
profile of DT40 is rather similar to that of bursal B cells [79]. Avian B cell maturation occurs in a specific organ – bursa of Fabricius, where B cells diversify their Ig genes by somatic gene conversion [80], a process that continues at high rate in DT40 cells [77, 78]. In many respects bursal B cells also resemble GC B cells, as the bursal B cells proliferate [81], diversify their Ig repertoire [80], undergo selection events involving apoptosis [82], and express common genes such as Bcl-6 [79] and activationinduced cytidine deaminase (AID) [83, 84], which is indispensable for both SHM [85] and gene conversion [83, 84]. The high homologous recombination frequency of DT40 cell line makes it an attractive model for gene targeting [86], and DT40 cells have been used successfully in several studies of transcriptional regulators [87]. Inactivation of both Pax5-alleles in the DT40 cell line leads to the loss of functional B cell identity [18], in line with the results from conditionally Pax5-deficient mature mouse B cells [13]. However, Pax5-deficient DT40 cells exhibit a clear transition towards the plasma cell state, as they have diminished Bcl-6 expression with simultaneous upregulation of Blimp-1 and XBP-1 accompanied with substantially elevated secretion of soluble IgM into the supernatant [18]. As restored Pax5 expression is able to normalize the altered expression of Bcl-6, Blimp-1 and XBP-1 in Pax5)/) DT40 cells [18], the plasma cell characteristic expression of these factors is indeed a consequence of Pax5 deficiency. Pax5 has been considered to repress XBP-1 expression [15], which is also supported by studies with Pax5-deficient DT40 cells [18]. However, despite the fact that restored Pax5 expression downregulates XBP-1 expression
close to the level of wild-type cells, the Pax5)/) cells with enforced Pax5 expression still secrete more IgM into the supernatant than the wild-type cells [18]. Considering that XBP-1 mRNA is spliced in response to ER stress to produce the shorter form that promotes Ig secretion [53, 58], and that both Pax5-deficient as well as the Pax5)/) cells with restored Pax5 expression predominantly express the spliced form of XBP-1 [18], it is evident that Pax5 has no effect on XBP-1 splicing. Given that in DT40 cells both unspliced and spliced XBP-1 isoforms are normally found at low basal level [18], it is possible that the inactivation of Pax5 originally increases the amount of both isoforms, which then leads to increased secretion of Ig and consequent stress in ER, hence promoting further XBP-1 splicing. These events could explain why restored Pax5 expression does not suppress Ig secretion in Pax5)/) DT40 cells [18]. Although XBP-1 expression is downregulated, the continuous Ig secretion ensures that the expressed XPB-1 remains to be spliced regardless of restored expression of Pax5. Nevertheless, Pax5 appears to repress XBP-1 expression [15, 18], but does not contribute to the regulation of XBP-1 splicing [18]. Blimp-1 is a target for repression by Bcl-6 [11, 16, 17], whereas the suppression of Bcl-6 expression is mediated by Blimp-1 [6]. The fact that Bcl-6 is downregulated and Blimp-1 upregulated due to the loss of Pax5 function in DT40 cells [18] leads to an intriguing question as to whether repression of Bcl-6 or upregulation of Blimp-1 constitutes the primary event when Pax5 expression is lost. According to transfection studies, Pax5 is probably needed for the sustained
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expression of Bcl-6 rather than for the inhibition of Blimp-1, as ectopic expression of Bcl-6 in the Pax5)/) DT40 cells is sufficient to block the Blimp-1 induction [18], thus suggesting that Pax5 is not an essential inhibitor of Blimp-1. However, Delogu et al. described that loss of Pax5 leads to the activation of plasma cell genes [19]. In their studies, conditional Pax5 inactivation in mouse B cells promoted reactivation of Pax5repressed genes including J-chain, Cd28 and Ccr2, which are important for plasma cell differentiation [19, 88]. Moreover, loss of Pax5 in mature mouse B cells was shown to activate the Prdm1 gene encoding Blimp-1 [19], suggesting that Pax5 may also suppress the expression of Blimp-1. In contrast to our studies with Pax5-deficient DT40 cells [18], Delogu et al. did not find an altered expression of Bcl-6 or XBP-1 in the conditionally Pax5-deficient mouse B cells [19]. This is surprising, considering that Blimp-1 expression appears to promote the downregulation of Bcl-6 [6, 18], and transfection studies in DT40 cell line show that Pax5 is not demanded for Blimp-1 suppression [18]. However, the fact that Pax5 is not essential for the inhibition of Blimp-1 does not exclude the possibility that it could also have a role in Blimp-1 repression. Although recent studies with Pax5-deficient avian [18] and murine [19] B cells both support the idea that Pax5 is an essential inhibitor of plasma cell differentiation, and is downregulated during the antigen-driven B cell activation, the two experimental models differ considerably. This discrepancy might be related to the fact that, in many respects, DT40 cells reflect an activated GC B cell state, thus resulting in the more profound plasma cell phenotype and Ig secretion upon Pax5 inactivation [18]. However, the possibility of a species difference between the avian and murine systems in the regulation of B cell activation cannot be excluded. On the other hand, DT40 cells overexpress c-myc [89], a key regulator of cell proliferation, which is thought to be downregulated by Blimp-1 after the proliferative steps of B cell activation [90]. Furthermore, enforced Blimp-1 expression has been shown to induce apoptosis in the only partially activated B cells [91], and conditional Pax5 inactivation in mice leads to the loss of mature B cell population [13]. Therefore, as the inactivation of Pax5 promotes spontaneous Blimp-1 activation [18, 19], it remains possible that conditionally Pax5-deficient murine B cells [13, 19] are depleted due to the premature Blimp-1 induction before they reach the Ig secreting stage observed in Pax5)/) DT40 cells [18]. Nevertheless, despite the differences in the Pax5-deficient experimental systems in avian [18] and murine [19], both systems clearly demonstrate the continuous requirement for Pax5 in the maintenance of B cell identity and prevention of premature plasma cell differentiation.
Pax5 controls B cell affinity maturation by regulating the expression of AID and Aiolos B cell affinity maturation occurs in GCs [2, 3], where SHM and CSR are central events diversifying rearranged Ig genes to produce different isotypes of high-affinity antibodies (Fig. 1). Activated high-affinity B cells are produced during iterative cycles of proliferation [63], SHM [63, 64] and selection [65] with the CSR yielding antibodies of different Ig isotypes [2, 3]. Both SHM and CSR are critically controlled by AID [85, 92, 93], and the expression of AID is restricted to GCs [94]. However, AID is also expressed in DT40 cells [18, 83, 84], where it is needed for Ig gene conversion [83, 84]. The putative regulatory region of AID contains a Pax5 binding site, which is indispensable for AID expression [95]. Moreover, Pax5-deficient DT40 cells fail to express AID [18], while enforced Pax5 expression can induce AID expression in a pro-B cell line [95]. Given that ectopic Pax5 overexpression in the Pax5)/) DT40 cells results in the overexpression of AID [18], Pax5 appears to directly activate AID expression. Furthermore, enforced expression of Blimp-1 in B cell lines or splenic mouse B cells has been shown to cause AID downregulation [6], but as AID remains to be overexpressed in Pax5)/) DT40 cells ectopically expressing both Blimp-1 and Pax5 [18], it seems that Blimp-1 may suppress AID expression via repression of Pax5. In addition to AID [94], GC B cells also express high amounts of Bcl-6 [69, 70], which is considered to be critical for the affinity maturation [1–3] due to its role in both GC formation [66–68] and prevention of premature Blimp-1 induction [11, 16, 17]. By regulating GC formation and Blimp-1 expression, Bcl-6 also indirectly contributes to AID expression, although the fact that AID is not expressed in Pax5-deficient DT40 cells where Bcl-6 expression is restored [18] clearly indicates that Bcl-6 does not promote AID expression directly. On the other hand, as Bcl-6 expression depends on Pax5 [18], most likely Pax5 has a crucial role in the regulation of GC reaction, thus contributing to affinity maturation. The expression of transcription factor Aiolos is dependent on Pax5 [18]. The Ikaros family member Aiolos sets the threshold for B cell activation [96]. Aiolos null mutant mice are characterized by spontaneous GC formation as well as elevated serum IgG and IgE levels without immunization [97], hence indicating a role for Aiolos in the regulation of GC formation and function. Moreover, Aiolos-deficient mice fail to generate highaffinity bone marrow plasma cells due to a B cell intrinsic defect [98]. The amount of Aiolos transcripts is diminished in Pax5)/) DT40 cells, where Pax5 overexpression results in the increased transcription of Aiolos [18], thus demonstrating that Pax5 regulates Aiolos expression. Interestingly, the expression of EBF, thought 2006 The Authors
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to act upstream of Pax5 [21–24], is also regulated by Pax5 [18]. In contrast, Pax5 does not regulate the expression of Ikaros [18], a critical regulator of BCR signalling [99] and early B cell development [21, 22]. Nevertheless, the evidence indicating that Pax5 has a critical role in the regulation of AID, Aiolos and Bcl-6 [18], strongly supports the idea that Pax5 is needed for the affinity maturation of B cells during GC reaction. Furthermore, considering that Aiolos is indispensable for the generation of high-affinity bone marrow plasma cells, Pax5 appears to have importance for long-term immunity and memory.
The versatile role of Pax5 during B cell activation Pax5 is expressed throughout the B cell lineage [29, 30], where it ensures B cell commitment [25–28] and maintains B cell identity [13, 14] by suppressing the expression of lineage-inappropriate genes [21, 26, 33] while simultaneously promoting the expression of B cell-specific genes [21, 36–39]. Before B cell activation, Pax5 is thought to contribute to the sustained expression of BCR and its signalling machinery, as expression of sIgM and several cytoplasmic BCR signalling molecules is downregulated in the absence of Pax5 [13, 18, 38]. Considering that BCR-mediated antigen recognition constitutes a key element in B cell activation, the importance of Pax5 function prior to B cell activation is evident. Following B cell activation, Pax5 expression is suppressed [12, 19, 29] resulting in the downregulation of B cell-specific genes [18, 19] and the induction of previously repressed genes [19] as well as plasma cell-specific genes [18, 19]. One of the plasma cell specific proteins expressed in the absence of Pax5 is Blimp-1 [18, 19], which is thought to directly repress Pax5 expression during plasma cell differentiation [12]. The fact that depletion of Pax5 from B cells results in strong Blimp-1 induction [19] suggests that a reciprocal inhibition mechanism exists between Pax5 and Blimp-1. However, Pax5 is not essential for repression of Blimp-1, as Bcl-6 expression is sufficient for the downregulation of Blimp-1 in the absence of Pax5 [18]. On the other hand, Bcl-6 appears to be an important inhibitor of plasma cell phenotype only in GC B cells [69, 70], and Bcl-6 downregulation was not observed upon conditional inactivation of Pax5 from mature mouse B cells [19]. Therefore, it is possible that the altered gene expression pattern observed in the conditionally Pax5-deficient mouse B cells [19] represents direct primary plasma cell differentiation rather than GC reaction. Considering that activated B cells proliferate during the initial stages of plasma cell differentiation [1–3], and multiple factors promoting the proliferation are repressed by Blimp-1 in plasmacytic cells [6, 90], Pax5 is needed to ensure sufficient proliferation via Blimp-1 inhibition. Accordingly, overexpression
of Pax5 in splenic B cells has been shown to promote proliferation [100], whereas loss of Pax5 seems to slow the proliferation [18, 100]. The role of proliferation during B cell activation is essential, and enforced Blimp-1 expression induces apoptosis in partially activated B cells [91]. Therefore, the Pax5-mediated repression of Blimp-1 induction [18, 19] has great importance in the early proliferative stages of plasmacytic differentiation. In activated GC B cells, Bcl-6 is thought to be responsible for the suppression of premature Blimp-1 induction [16, 17], supported also by the fact that Bcl-6 is sufficient to downregulate Blimp-1 expression in the absence of Pax5 [18]. In DT40 cells reflecting the activated GC B cell state, the loss of Pax5 results in diminished Bcl-6 expression [18], showing that Pax5 is needed to maintain the Bcl-6 expression during GC reaction (Fig. 2). This is further supported by the fact that enforced Pax5 expression causes the overexpression of Bcl-6 in the Pax5)/) DT40 cells [18]. During GC reaction, AID expression is crucial for Ig affinity maturation and CSR [85, 94], both of which also require continuous Pax5 expression, as Pax5 regulates the expression of AID [18, 95]. Moreover, Pax5 also prevents the premature Ig secretion by repressing the expression of IgH [49], J-chain [19, 50–52] and XBP-1 [15, 18]. Therefore, the biological significance of Pax5 is to first allow sufficient clonal expansion accompanied by AID-mediated CSR and Ig gene diversification. When B cells with high affinity for antigen are selected, Pax5 is downregulated to promote the terminal differentiation into plasma cells. Both Pax5 and Bcl-6 are alone sufficient to prevent plasma cell differentiation [9–12]. Conversely, the loss of either Pax5 or Bcl-6 results in Blimp-1 induction [17– 19], which drives plasmacytic differentiation [4, 7]. In theory, downregulation of either Pax5 or Bcl-6 from GC B cells would promote plasma cell differentiation, thus adding plasticity to the regulatory pathway of terminal B cell differentiation. Furthermore, the balance between the protein levels of Pax5 and Bcl-6 most likely serves an important function in the differentiation of activated B cells to produce memory B cells and plasma cells. Nevertheless, it remains to be established whether Pax5 or Bcl-6 is initially downregulated in GC B cells to allow plasmacytic differentiation.
Concluding remarks Recent findings have provided insight into the functional importance of Pax5 during the late events of B cell differentiation. As a consequence, the role of Pax5 has extended from early commitment and maintenance factor to a critical inhibitor of plasma cell fate. According to current understanding, Pax5 expression is required continuously throughout the B cell lineage to maintain functional B cell identity and to repress the premature induction of
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Figure 2 A model illustrating the versatile role of Pax5 in the inhibition of plasma cell differentiation. In mature resting B cells, Pax5 maintains functional B cell identity by activating B cell-specific genes and repressing the lineage-inappropriate genes. At the same time, Pax5 represses the expression of plasmacytic transcription factors Blimp-1 and XBP-1. In activated germinal centre (GC) B cells, Pax5 also maintains the adequate level of Bcl-6, thus contributing to Blimp-1 inhibition. The concerted action of Pax5 and Bcl-6 prevents premature Blimp-1 induction allowing proliferation and affinity maturation mediated by Pax5-induced expression of AID. Pax5 also regulates GC formation and function by maintaining the expression of Aiolos. In plasma cells, Blimp-1-mediated downregulation of Pax5 and Bcl-6 leads to irreversible terminal differentiation. In the absence of Pax5, plasmacytic cells lose their B cell identity, and upregulation of XBP-1 promotes the increased immunoglobulin secretion.
plasma cell programme. During the early stages of B cell development the induction of Pax5 expression leads to irreversible commitment to B cell lineage, whereas upon antigen-driven B cell activation the downregulation of Pax5 results in the loss of B cell identity and terminal differentiation into plasma cells. Considering the critical importance of Pax5 in the maintenance of Bcl-6 as well as in the suppression of Blimp-1 induction, the downregulation of Pax5 is probably involved when the onset of late Blimp-1-mediated plasma cell differentiation is triggered. However, the molecular regulatory mechanisms guiding these events are still poorly understood and should be elucidated by further exploration.
Acknowledgments This work was financially supported by The European Union (QLK3-CT-2000-00785), Tekes (the National Technology Agency), the Academy of Finland, Turku Graduate School of Biomedical Sciences, the Finnish Cultural Foundation, the Finnish Cultural Foundation of Southwest Finland, Juliana von Wendt Foundation, Turku University Foundation, the Finnish Cancer Union, Emil and Blida Maunula Foundation, Paulo Foundation, Emil Aaltonen Foundation and Ida Montin Foundation.
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