Calmodulin Inhibition of E2A Differentiation Into Plasma Cells by Initiation of Antigen Receptor-Dependent

Home , ADD3, BACH2, C1QBP, Enolase 2, ETS1, FARS2

The Journal of Immunology

Initiation of Antigen Receptor-Dependent Differentiation into Plasma Cells by Calmodulin Inhibition of E2A1

Jannek Hauser, Jiyoti Verma-Gaur, Anders Wallenius, and Thomas Grundstro¨m2

Differentiation of B lymphocytes into Ab-secreting plasmablasts and plasma cells is Ag driven. The interaction of Ag with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. However, not much is known about the coupling between BCR activation and the shift in transcription factor network from that of a B cell to that of ASC differentiation. Our genome-wide analysis shows that Ab-secreting cell differentiation of mouse B cells is induced by BCR activation through very fast regulatory events from the BCR. We identify activation of IFN regulatory factor-4 and down- regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as immediate early events. Furthermore, the transcription factor E2A is required for the rapid key down-regulations after BCR activation, and the Ca2؉ sensor protein calmodulin has the corresponding regulatory effect as BCR activation. Moreover, mutants in the calmodulin binding site of E2A show that Ca2؉ signaling through calmodulin inhibition of E2A is essential for the rapid down-regulation of immediate early genes after BCR activation in initiation of plasma cell differentiation. The Journal of Immunology, 2009, 183: 1179–1187.

hen encountering Ags, B lymphocytes can adapt to tion program (1, 3, 7). Pax5 is essential to establish and maintain produce a highly specific and potent Ab response. B cell identity. It activates many B lineage-specific genes and si- The affinity maturation of Abs and differentiation of B multaneously represses many genes associated with stem cell and W 3 cells into Ab-secreting cells (ASC) are crucial components of the non-B lineage programs as well as a number of genes involved in immune response. B cells that successfully respond to Ag can dif- ASC differentiation, including Prdm1, the gene encoding B lym- ferentiate into plasmablasts (short-lived proliferating ASC), into phocyte-induced maturation protein 1 (Blimp1), and Xbp-1. long-lived noncycling Ab-secreting plasma cells, and into memory Therefore, Pax5 must be repressed to allow ASC differentiation B cells that can rapidly differentiate into ASC after re-exposure to (1–3). Bcl-6, Ets-1, Spi-B, and BACH2 are also suppressors of Ag (1). It is well established that plasma cell development is Ag ASC development, in part by repressing Blimp1 and Xbp-1 (1, 3, driven (1–3): signaling from Ag receptors (BCRs) with their mem- 7, 8). In addition, MITF represses IFN regulatory factor (IRF)-4, brane-bound Abs plays a key role in the start of ASC differenti- another key regulator of the ASC development (1, 3, 9). In ASC, ation. The BCR is a fundamental determinant of early plasma cell however, IRF-4 and Blimp1 inhibit Pax5 and Bcl-6 (1, 3, 9, 10). differentiation (4), and the strength of the BCR interaction with Ag The Fli-1 transcription factor has recently been implicated in plays a critical role in determining which clones enter both early development and maintenance of B cells (11). The existence of extrafollicular plasma cell differentiation and plasma cell differen- these two different gene-regulatory transcription factor net- tiation after successful affinity maturation in the germinal center works based on mutual repression has been established mainly (4–6). Only germinal center B cells that have acquired high af- by studies that examine the effects of overexpression or loss of finity for the immunizing Ag form plasma cells. Thereby, only expression of the different transcription factors. However, not cells that can produce optimal Ab specificities become plasma much is known about the coupling between BCR activation and cells, which ensures effective immunity (4, 6). the shift in transcription factor network from that of a B cell to The developmental program leading to ASC differentiation from that of ASC differentiation. B cells is controlled by a gene-regulatory network in which tran- In this study, we report that ASC differentiation of mouse B scriptional repression plays a key role (1). A small group of tran- cells is induced by BCR activation through very fast regulatory scription factors expressed in the B cells, including Pax5, Bcl-6, events from the BCR. Primary regulatory events were identified as MITF, Ets-1, and BACH2, are repressors of the ASC differentia- inhibition of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as well as the activation of IRF-4 gene expression. We further show that Ca2ϩ signaling through inhibition of E2A by the Department of Molecular Biology, Umeå University, Umeå, Sweden Ca2ϩ sensor calmodulin (CaM) is essential for the rapid down- Received for publication February 11, 2009. Accepted for publication May 5, 2009. regulation of immediate early genes after BCR activation in initi- The costs of publication of this article were defrayed in part by the payment of page ation of plasma cell differentiation. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Swedish Research Council and the Materials and Methods Swedish Cancer Society. Activation of B lymphocytes from mouse spleens 2 Address correspondence and reprint requests to Dr. Thomas Grundstro¨m, Depart- ment of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden. E-mail Primary B lymphocytes were purified from mouse spleens and main- address: [email protected] tained as previously described (12), and stimulated with LPS (Calbio- chem), IL-4 (PeproTech), and/or CD40L (R&D Systems), as indicated, 3 Abbreviations used in this paper: ASC, Ab-secreting cell; Blimp1, B lymphocyte- at 10 ␮g/ml, 5 ng/ml, and 200 ng/ml, respectively, unless otherwise induced maturation protein 1; CaM, calmodulin; CaMK, CaM-dependent protein ki- 6 nase; IRF, IFN regulatory factor; MSCV, murine stem cell virus; PKC, protein kinase indicated. The BCR of stimulated cells at 10 cells/ml was (where in- Ј C; shRNA, short hairpin RNA. dicated) activated by incubation with goat F(ab )2 anti-mouse IgM (Southern Biotechnology Associates) at 2.5 ␮g/ml (5 ␮g/ml for Figs. 1 Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 and 6) for the indicated times. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900455 1180 INITIATION OF DIFFERENTIATION TO PLASMA CELLS

DNA microarray analysis The analysis of gene expression changes in B cells upon BCR activation was performed using the Illumina BeadChip system. For in vitro transcription amplification, 200 ng of RNA was used with the Illumina RNA Am- plification Kit (Ambion). Amplified RNA (1.5 ␮g) was hybridized to the Sentrix MouseRef-6 Expression Beadchip (Illumina) containing 47,667 probes. The primary data were collected from the BeadChips using the manufacturer’s BeadArray Reader and analyzed using the supplied scanner software. Data normalization was performed by cubic spline normalization using Illumina’s Beadstudio v3 software. Clustering of certain selected genes was done using cluster 3 software, and data were visualized using tree view v1.2 software. All raw CEL dataset files are available at www. ncbi.nlm.nih.gov/geo (accession no. GSE15606).

Western blot Nuclear extracts were prepared, as previously described (13), and Western blot was performed using the WesternBreeze immunodetection system (In- vitrogen), according to the manufacturer’s instructions, and using Abs listed in supplemental Materials and Methods.4

Plasmids and viruses The pcDNAI-based expression vector for mammalian CaM, the short hairpin RNA (shRNA) that interferes with E12/E47-speciﬁc human RNA, the EBV-based shuttle vector pMEP4 derivatives encoding wild-type and CaM-resistant m847 and m8N47 mutants of mouse E12, and the murine stem cell virus (MSCV)-internal ribosome entry site-GFP-based retrovi- ruses have all been described previously (12–14).

Infection of mouse B lymphocytes Retrovirus concentrated by centrifugation was added with 5 ␮g/ml poly- brene to 0.5 ϫ 106 puriﬁed B cells after activation with LPS plus IL-4 for 14 h (24 h in Fig. 6). After a 12-h incubation, the infection was repeated for 12 h, followed by incubation for a further 22 h postinfection in fresh complete medium with the stimulants to allow for expression of GFP and E12 or CaM. In experiments after activation with CD40L plus IL-4, the medium was supplemented with LPS (2.5 ␮g/ml) during retroviral infection incubations to improve infection efﬁciency. Where indicated, anti- mouse IgM was added for the indicated time. Intracellular immunostaining of harvested cells was done with 2% paraformaldehyde and ethanol, as previously described (15), using Abs as listed in Materials and Methods. Stainings were done for at least 30 min at room temperature in the dark. Flow cytometry was with a FACSCalibur instrument and analysis with CellQuest software (BD Biosciences).

Cell culture and transfections The human B cell lymphoma line DG75 (16) was maintained in RPMI FIGURE 1. Differentiation of activated B lymphocytes from mouse 1640 medium supplemented with 5% FCS and antibiotics. DG75 cells were transfected by electroporation with 2 ␮g of CaM expression plasmid or the spleen to plasma cells is Ag receptor driven. Cells were maintained with empty pcDNAI/amp vector. Eight hours later, live cells were separated LPS plus IL-4 activators (A and B) or CD40L plus IL-4 activators (C and using Lymphoprep (Axis-Shield), and 12 h later anti-IgM was added to D) at the indicated concentrations with or without addition of anti-IgM one-half of the cells, followed by continued culture for 3 h. DG75 cells after 2 days of culture. Cells were harvested 1 or 2 days later, as indicated, transfected to express empty pMEP4 derivative, or pMEP4 derivative en- immunostained against syndecan-1 (CD138), and analyzed by FACS. Per- coding the shRNA, E12, or m847 or m8N47 mutant of E12, were selected centages of total analyzed cells are given in the upper quadrants. with hygromycin B (Roche) for 5 days, as described previously (12). The BCR of DG75 was activated by stimulation of 1 ϫ 107 cells for 3 h, unless otherwise specified, with 5 ␮g of goat F(abЈ) anti-human IgM (Southern 2 Results Biotechnology Associates) in 1 ml of complete RPMI 1640 medium supplemented with 5% FCS. Very fast gene-regulatory events upon activation of the BCR To analyze whether ASC differentiation is Ag driven in an in vitro Real-time RT-PCR cultivation system, B cells purified from mouse spleen were first Total RNA was extracted using TRIzol reagent (Invitrogen), and real-time activated using LPS and IL-4, and the effect of activation of the Ag PCR analysis was performed, as previously described (12), using GAPDH receptor with anti-IgM after 2 days of cultivation was analyzed. To as an internal control. The specificity of the real-time PCR was analyzed by examine ASC differentiation, FACS analyses were performed after melt-curve analysis, as described by the manufacturer, and the sizes of the immunostaining for syndecan-1 (CD138), a marker for plasma- PCR products with the different primer pairs were verified by 2.5% agarose gel electrophoresis. Some of the primer pairs used have been described blasts and plasma cells. Compared with the cells not given BCR previously (12), and the additional RT-PCR primer pairs used are listed in activation, BCR activation for 1 day produced many more plas- supplemental Materials and Methods.4 mablasts/plasma cells, identified by increased expression of syndecan-1 (Fig. 1A). Furthermore, compared with cells not treated with anti-IgM, the average level of syndecan-1 expression in- 4 The online version of this article contains supplemental material. creased between 2.5 and 3 times in this increased population of The Journal of Immunology 1181 highly syndecan-1-expressing cells. After a second day of BCR activation, the highly syndecan-1-expressing cells increased in size, a change expected for differentiation into ASC cells. In the control without BCR activation, the frequencies of large and strongly syndecan-1-expressing cells changed little (Fig. 1A). Thus, activation of the Ag receptor increases plasmablast/plasma cell differentiation in this system. The increase in frequency of syndecan-1 high cells and their syndecan-1 expression level at day 1 and the shift to larger highly syndecan-1-expressing cells at day 2 were also obtained when stimulating the BCR of cells activated with 4-fold lower levels of LPS and IL-4 (Fig. 1B). In the germinal center, the Ag receptor works with Th cells to determine selection of B cells for plasma cell differentiation, and CD40L (CD154) is the most important cytokine from the Th cells (2, 17). Therefore, we analyzed the effect of activation of the BCR with anti-IgM also when cultivating the splenic mouse B cells with CD40L instead of LPS together with the IL-4. This condition also resulted in a profound increase in large and highly syndecan-1- expressing cells when the BCR was stimulated (Fig. 1C). The increase in large and highly syndecan-1-expressing cells by stimulating the BCR was even stronger when the levels of CD40L and IL-4 were reduced 4- or 10-fold (Fig. 1D, and data not shown). Thus, Ag receptor stimulation increases plasmablast/plasma cell differentiation both in Th cell-independent and Th cell-dependent differentiation and over a range of concentrations of LPS, CD40L, and IL-4. BCR activation results in a large number of very rapid transcription changes To search for rapid changes in gene expression following BCR activation, we performed DNA microarray analysis of activated splenic B cells with and without anti-IgM treatment for 3 h. Cells activated with CD40L and IL-4 were used because this activation resulted in the largest increase in ASC differentiation (cf Fig. 1, A FIGURE 2. Rapid differential gene expression upon BCR activation. and B with Fig. 1, C and D) and because most Ab responses are Th Cluster plots of selected important genes differentially expressed during 3 h cell dependent, mimicked by the main cytokine CD40L. The of anti-IgM stimulation of the BCR of B lymphocytes from mouse spleen activated with CD40L (200 ng/ml) plus IL-4 (5 ng/ml). Total RNA was change in expression for each gene was computed as a ratio of isolated from B cells of three independent mice with and without the stim- expression in anti-IgM-treated B cells vs untreated control B cells. ulation and amplified and hybridized on Illumina BeadChips. The fold Using the Illumina Mouse Beadchip system, the expression of differential expression of genes in the individual mice is color coded, with 31,492 genes was examined. Dendrogram analysis of these genes increases marked in red and decreases marked as green. The functional demonstrated a high degree of reproducibility between the three classification of genes was done using gene ontology information provided mice analyzed (data not shown). The expression of a remarkably by Illumina, and selected genes are included. large set of genes differed significantly (Ն1.5-fold change, p Ͻ 0.05) between the BCR-stimulated and nonstimulated B cells: 2,259 genes were up-regulated, and 2,345 genes were down-reg- which has been correlated with the commitment to the plasma cell ulated. The cluster plots of certain genes with a coupling to B cell lineage (18). The finding that ϳ7% of the genes were up-regulated and/or ASC differentiation are shown in Fig. 2, and the complete and 7% were down-regulated within3hofBCRactivation (sup- result of BCR activation for the 4,604 genes with significantly plemental Table S1)4 is comparable to the reported difference in changed expression is shown in supplemental Table I.4 The anal- expression of 15% of the genes (Ն2-fold) between purified mouse ysis reveals that BCR activation, which promotes terminal differ- germinal center B cells and plasma cells (19). Thus, a significant entiation of B cells, reduces expression within3hofmajor classes part of the change in mRNA level happens already within3hfor of genes that are highly expressed in primary and germinal center most of the genes that change expression in differentiation from a B cells, including genes important for B cell identity, such as se- B cell to an ASC. cretory factors (e.g., IL-16 and Ltb), surface proteins (e.g., CD22 To study the earliest Ag receptor-driven transcriptional changes and Btla), and transcription factors (e.g., Bcl-6, Pax5, Oct-2, in splenic B cells, the mRNA levels were followed by quantitative BACH2, Ets-1, MTA3, and Spi-B) (Fig. 2). The levels of the de- real-time PCR after the BCR was stimulated for selected genes creases in expression of the transcription factors within 3 h varied identified in our genome-wide microarray analysis (Fig. 2) and for from ϳ2-fold to a 10-fold down-regulation for Ets-1. certain genes previously reported to change expression during Plasmacytic differentiation requires the induction of several ASC differentiation or to have a role in this process (1–3, 20). transcription factors that are important for this development, and First, cells preactivated with LPS and IL-4 were analyzed (Fig. one of these is IRF-4. Interestingly, IRF-4 showed more than a 3A). The mRNA levels did not change significantly, or changed 3-fold up-regulation within3hofBCRactivation (Fig. 2 and only relatively slowly, during 12 h of activation of the BCR for supplemental Table S1).4 We found also that the BCR activation many of the proteins, including IgH, XBP-1, MTA3, BACH2, increased expression of many surface proteins, including CD27, IRF-8, STAT6, EBF, BHLH B3, and Aiolos (Fig. 3A, and data not 1182 INITIATION OF DIFFERENTIATION TO PLASMA CELLS

FIGURE 3. Changes in expression of different mRNA and proteins in B lymphocytes from mouse spleen activated with LPS plus IL-4 activators (A and B) or CD40L plus IL-4 activators (C) for 48 h, followed by stimulation of the BCR by addition of anti-IgM for the indicated times. A, The effects of BCR activation on the expression of the indicated mRNA, determined by quantitative RT-PCR and normal- ized using the mRNA levels of GAPDH. The levels before addition of anti-IgM were set at 100%. Results are mean Ϯ SD (n ϭ 3). B, Represen- tative Western blots from determina- tions of time courses of BCR-induced changes in levels of the indicated proteins with ␣-tubulin used as loading control. C, Changes in expression of the indicated mRNA in B lymphocytes from mouse spleen activated with CD40L and IL-4 for 48 h, followed by stimulation of the BCR by addition of anti-IgM for the indicated times. The mRNA levels were determined by quantitative RT-PCR, as in A (n ϭ 3).

shown). However, this was not the case for all genes. The mRNA of ASC differentiation are very early regulatory events that are initi- level for Pax5, a key transcription factor for B cell identity and ated within less than 30 min and carried far within 1 h when activating repressor of the ASC differentiation program, was significantly the Ag receptor of splenic B cells. The mRNA levels for IRF-4 and reduced already within 30 min of BCR stimulation. The reduction Blimp1, important regulators of ASC differentiation and repressors of was ϳ3-fold within 1 h and 4- to 5-fold within 3 h (Fig. 3A). The Pax5 and Bcl-6, increased relatively fast, although perhaps slightly mRNA level of Bcl-6, another repressor of ASC differentiation, slower than the six down-regulations. These mRNA increases reached also decreased significantly within 30 min of BCR stimulation, and 2-fold within less than 2 h (Fig. 3A). this decrease was also ϳ3-fold within 1 h and 4- to 5-fold within The very fast reductions of the mRNA levels for several repres- 3 h (Fig. 3A). The level of mRNA for MITF, one more repressor sors of ASC differentiation could be through inhibition of the tran- of ASC differentiation, was reduced by ϳ50% within 30 min of the scription, provided that the mRNA is constitutively short-lived, or, BCR stimulation (Fig. 3A). The mRNA levels of the Ets family alternatively, through anti-IgM-induced degradation of the mRNA. members Ets-1, Fli-1, and Spi-B, all implicated in maintenance of Therefore, the effect of BCR activation was compared with that of B cells (1, 3, 7, 8, 11), were also significantly reduced within 30 an inhibitor of transcription, actinomycin D. The mRNA level for min of the BCR stimulation (Fig. 3A). Their mRNA levels were each of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B was found to reduced between 2 and 3 times within 1 h, and they decreased fall with approximately the same rate as after anti-IgM treatment further within 3 h (Fig. 3A). The reduction was especially pro- also after actinomycin D treatment (data not shown). Thus, the nounced for Ets-1, which had only 18 Ϯ 6% of the mRNA remaining mRNAs of these genes are all constitutively short-lived, and the after 3 h. Thus, down-regulation of the mRNA for several repressors fast reduction in mRNA level after Ag receptor stimulation The Journal of Immunology 1183 appears to be through inhibition of transcription for each of these bisindolylmaleimide had little effect compared with Ca2ϩ chelator or genes. To determine whether the rapid reductions in the mRNA of Ca2ϩ channel blockers on the reductions in Pax5, Bcl-6, MITF, and these genes and the increase in Blimp1 mRNA after BCR activa- Ets-1 mRNA and the increase in Blimp1 mRNA after BCR stimulation resulted in corresponding changes at the protein level, we tion, whereas Fli-1 and Spi-B reductions and IRF-4 induction were performed Western blot analyses. A typical Western for each pro- affected by both types of treatment (supplemental Fig. S1).4 tein is shown in Fig. 3B. All reductions in mRNA levels led also Both stimulated splenic B cells and the easily manipulated human to reductions in the protein levels, although somewhat delayed B cell lymphoma line DG75 (16) were used to investigate the Ca2ϩ compared with the mRNA levels. At 30 min, when the mRNA signaling-dependent effects of BCR activation. As in stimulated levels were already reduced (Fig. 3A), the Pax5, Bcl-6, MITF, splenic B cells, activation of the BCR with anti-IgM rapidly reduced Ϯ Ϯ Ets-1, and Fli-1 protein levels remained at 105 10, 90 21, the levels of Pax5, Bcl-6, Ets-1, and Fli-1 mRNA and increased the Ϯ Ϯ Ϯ Ն 100 15, 95 3, and 92 9%, respectively (n 3). However, level of Blimp1 mRNA in the B cell line (Fig. 4A). MITF mRNA Ϯ Ϯ Ϯ the levels of these proteins did decrease to 85 9, 76 27, 51 could not be detected in this cell line, and Spi-B expression was not Ϯ Ϯ Ϯ 8, 93 17, and 80 8%, respectively, after 1 h, and to 56 12, inhibited (data not shown). The cause of these defects in MITF and 62 Ϯ 18, 38 Ϯ 4, 73 Ϯ 7, and 48 Ϯ 5%, respectively, after2h(n Ն Spi-B is unknown, but they might be coupled to the lymphoma phe- 3). In contrast, the increase in Blimp1 protein was several hours notype of this cell line. The mechanism of IRF-4 induction was not delayed compared with the increase in Blimp1 mRNA, indicating ϩ analyzed because IRF-4 is induced by NF-␬B (2) and the main Ca2 - the presence of posttranscriptional regulation of the protein level sensor protein CaM and CaMK II are essential for NF-␬B activation after the BCR activation for this protein. after Ag receptor activation (14, 22). We analyzed the time courses of the effects of activation of the BCR also after combining the IL-4 treatment of the splenic mouse B To examine whether CaM could affect the expression of the Pax5, cells with CD40L instead of LPS. The BCR stimulation resulted in the Bcl-6, MITF, Ets-1, Fli-1, and Blimp1 genes, the effects of its over- corresponding down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, expression were investigated. The efficiency of the CaM overexpres- and Spi-B mRNA, as well as a reduction in MTA3 and BACH2 and sion system in DG75 cells has been verified previously by Western up-regulation of IRF-4 and Blimp1 mRNA when using CD40L in- blot analysis (12). The overexpression of CaM reduced the Pax5, ϳ stead of LPS in the activation (Fig. 3C). Notably, the decrease in Bcl-6, and Fli-1 mRNA levels 2-fold and the Ets-1 mRNA level Bcl-6 mRNA was more pronounced when using CD40L. This agrees ϳ3-fold compared with vector control, and CaM increased the level with our observation that CD40L increases Bcl-6 mRNA expression of Blimp1 mRNA ϳ2-fold (Fig. 4B). The effects of overexpression of to a higher level than LPS does (data not shown), and with Bcl-6 CaM were also analyzed in LPS- and IL-4-activated splenic B cells at being a strongly expressed master regulator of germinal centers whose the protein level by FACS. The cells were infected with retrovirus- formation depends on Th cells and CD40L (2). The effects of BCR expressing CaM, followed by an internal ribosome entry site and stimulation on the expression of the 10 genes were similar also after GFP. The FACS plots of a representative experiment with Pax5 are combined stimulation with CD40L and LPS (at 4-fold reduced levels) shown in supplemental Fig. S2A.4 The signals obtained with Pax5 Ab together with IL-4 (data not shown). Thus, Ag receptor stimulation were up to 100-fold higher than without primary Ab, and 30–60% of results in the rapid key regulatory changes both in Th cell-independent the cells were infected. Analysis of the FACS plots showed that CaM and Th cell-dependent ASC differentiation. overexpression not only reduced Pax5 of GFP-positive cells, but also had some effect on noninfected cells of the same plot when compared The early regulatory events upon activation of the BCR depend ϩ with cells not exposed to CaM-overexpressing cells. This reproduc- on Ca2 signaling and CaM ible finding is probably a bystander effect, i.e., that infected cells can Activation of Ag receptor leads to formation of a large complex of signal to their neighboring cells. It is notable that Ca2ϩ and MAPK proteins below the receptor that rapidly increases the intracellular signaling pathways, both of which regulate Pax5 and the other tran- 2ϩ 2ϩ Ca concentration and results in a combination of Ca signaling scriptional regulators studied in this work, are involved in production 2ϩ and a cascade of phosphorylations (21). To investigate whether Ca of bystander effects on neighboring cells (23). To avoid any influence signaling and/or serine protein kinases were essential for the effects of of bystander effects on the results, the effects of overexpressions on BCR stimulation on the expression of Pax5, Bcl-6, MITF, Ets-1, Pax-5 and the other regulators were compared with vector control Fli-1, IRF-4, Spi-B, and Blimp1 mRNA, the activation with anti-IgM infection and not with noninfected cells of the same FACS plot. The was performed in the presence of various inhibitors of the signaling results of all experiments are summarized in Fig. 4C. As seen in the pathways. The Ca2ϩ chelator BAPTA-AM completely blocked the figure, up to 2-fold decreases in Pax5, Bcl-6, and MITF and increase effect of BCR stimulation on the mRNA level of all the eight genes ϩ in Blimp1 were obtained upon BCR activation of uninfected control studied, and inhibition of L-type Ca2 channels with Nifedipine or ϩ samples, a finding that agrees with our Western blot results (Fig. 3B). inositol 1,4,5-triphosphate receptor Ca2 channels with TMB-8 par- The CaM overexpression system, previously verified by Western blot tially or completely blocked the effect, especially when combined (supplemental Fig. S1).4 Thus, Ca2ϩ signaling was essential for the analysis (12), reduced Pax5, Bcl-6, and MITF, and increased Blimp1 effect of BCR stimulation on the expression of mRNA for each of protein levels also in the primary splenic B cells (Fig. 4C). Effects of these transcription factors. However, the effects of inhibitors of Ca2ϩ/ overexpression of CaM were also analyzed after activation with 4 CaM-dependent protein kinase (CaMK) or Ca2ϩ/CaM-dependent CD40L and IL-4 (Fig. 4D and supplemental Fig. S2B), and this re- ϳ phosphatase calcineurin varied for the different proteins and were in sulted in 2-fold decreases of Bcl-6, Ets-1, and Fli-1 expression. The most cases smaller or absent (supplemental Fig. S1).4 The MAPK level of Bcl-6 protein was higher at this condition compared with in inhibitor PD98059 blocked the reductions in Pax5, Bcl-6, MITF, and presence of LPS plus IL-4, and the reductions in Bcl-6 protein on Spi-B mRNA and the increase in IRF-4 mRNA, and it partially CaM overexpression and after BCR activation were larger (Fig. 4, cf blocked the increase in Blimp1 mRNA and the reduction in Ets-1 and C and D), findings that agree with our observations that CD40L plus perhaps also Fli-1 mRNA (supplemental Fig. S1).4 This suggests that, IL-4 activate Bcl-6 mRNA much more than LPS plus IL-4 (data not in addition to the Ca2ϩ signaling, MAPK signaling is important to shown). In summary, Fig. 4, B–D, shows that overexpression of CaM different degrees for the BCR-induced changes in expression of all or has the corresponding effect as BCR activation on Pax5, Bcl-6, MITF, most of the analyzed genes. The protein kinase C (PKC) inhibitor Ets-1, Fli-1, and Blimp1 expression. 1184 INITIATION OF DIFFERENTIATION TO PLASMA CELLS

the BCR did not significantly affect the level of E2A mRNA for at least 12 h in mouse splenic B cells (supplemental Fig. S3).4 Id proteins, which are inhibitors of DNA binding of E proteins, participate at several regulatory steps in B cell lineage development (26, 27). However, none of the Id proteins showed a large increase in mRNA level during5hofBCRactivation that could explain the pronounced rapid decreases in Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B or the increase in Blimp1 mRNA in mouse splenic B lymphocytes (supplemental Fig. S3)4 or DG75 B cells (12). In addition, no significant change is seen in the expression level of any of the Id proteins in DG75 B cells (12). Furthermore, BCR activation does not decrease the amount of E2A that can bind to DNA in the absence of Ca2ϩ (12); therefore, no decrease in the level of E2A or increased inhibition of its DNA binding by Id proteins mediates the rapid transcriptional effects of BCR stimulation. We have reported a series of mutants in the basic DNA and CaM-binding sequence of the E2A isoform E12 that through com- binations of mutations are resistant to CaM to different extents (13). To investigate the possible role of Ca2ϩ/CaM inhibition of E2A in the regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Blimp1, we used the most CaM resistant of these, the m847 mutant, which has three amino acid substitutions. This mutant, however, is also resistant to Id proteins, whereas a second mutant used, m8N47, is sensitive to Id proteins and almost as resistant as the m847 mutant to CaM (12). We used a hygromycin-selectable EBV-based shuttle vector that stably directs synthesis of shRNA that interferes with human E2A mRNA. It reduces expression of both E2A mRNA and E2A protein over 10-fold in DG75 B cells (12). This shRNA expression plasmid reduced expression of Pax5 and Ets-1 mRNA in DG75 B cells by ϳ2-fold, Bcl-6 by ϳ1.3-fold, and Fli-1 by 3-fold, whereas Blimp1 expression was increased by ϳ2-fold (Fig. 5A), showing that the level of expression of these genes is E2A dependent. The reduction in E2A and the resulting FIGURE 4. Changes in expression of key proteins in plasma cell dif- effects on the expression of these genes were specific, because the ferentiation by CaM. A, Changes in expression of the indicated mRNA in shRNA expression plasmid did not affect the expression of a num- DG75 B cells following BCR stimulation with anti-IgM for the indicated ber of other BCR-regulated genes that have not been reported to be times. The levels were determined as in Fig. 3. B, Effects of CaM over- E2A regulated (Fig. 5A) (12) (data not shown). expression on the indicated mRNA levels with and without inhibition of BCR activation with anti-IgM reduced Pax5, Ets-1, Bcl-6, and the gene by BCR engagement. DG75 cells were transfected with 2 ␮gof Fli-1 mRNA 1.3- to 3-fold and increased Blimp1 mRNA 2- to CaM expression plasmid or the empty pcDNAI/amp vector (vec.), and anti-IgM treatments were for 3 h. The mRNA levels were determined as in Fig. 3-fold in nontransfected DG75 cells or in the presence of vector 3. C, Effects of CaM overexpression on the indicated protein levels in B lym- control plasmid (Fig. 5A). In contrast, expression of these genes phocytes from mouse spleen activated with LPS and IL-4 for 60 h, followed was approximately at the level of the anti-IgM-treated cells trans- by BCR stimulation with anti-IgM for 3 h (MITF), 5 h (Pax5 and Bcl-6), or fected with vector control both with and without anti-IgM treat- 12 h (Blimp1). Cells were infected with the empty MSCV retrovirus vector or ment when the shRNA expression plasmid was present (Fig. 5A). virus encoding CaM and GFP. The levels of the proteins were determined by Thus, BCR activation had no further effect on the expression of intracellular immunostaining and flow cytometry. Cells with a large increase in any of these genes when E2A expression was inhibited (Fig. 5A). GFP fluorescence were considered as infected and used to calculate protein This loss of effect of anti-IgM was specific, because expression of expression levels. The level in control cells infected with empty MSCV vector the shRNA against E2A did not influence the effect of anti-IgM for (vec.) and not treated with anti-IgM was set at 100%. D, Expression levels of any control gene analyzed that was induced or inhibited by BCR Bcl-6, Ets-1, and Fli-1 in corresponding CaM overexpression experiments, as in C, with anti-IgM treatments for5hofBlymphocytes activated with CD40L activation and not E2A regulated (Fig. 5A) (12) (data not shown). plus IL-4. All results are mean Ϯ SD (n Ն 3). The decrease in E2A mRNA was reversed by cotransfection with expression plasmid for the mouse E12 isoform of E2A, because the expressed shRNA does not interfere with mouse E2A. This complementation did not significantly change the expression Regulation of expression of repressors of plasma cell of any control gene, again confirming that they are not E protein differentiation and Blimp1 by the BCR depends on CaM regulated (Fig. 5A) (12) (data not shown). This complementation sensitivity of E2A fully restored both the levels of expression of Pax5, Bcl-6, Ets-1, Because Ca2ϩ-loaded CaM can inhibit DNA binding of E2A and Fli-1, and Blimp1 mRNA and the sensitivities to anti-IgM treat- other E proteins by directly binding to the DNA-binding basic ment (Fig. 5A). Importantly, the sensitivity to BCR stimulation sequences in their basic-helix-loop-helix domains (13, 24, 25), we was completely lost when complementing with a CaM-resistant examined whether E2A participates in the initiation of ASC dif- mutant of E12. Anti-IgM had no effect on expression of any of the ferentiation when BCR is activated. Previously, we have shown genes upon complementation with either the m847 or the m8N47 that BCR activation does not affect the level of E2A mRNA or mutant of E12 (Fig. 5A). The loss of the effects of BCR stimulation protein in DG75 B cells for several hours (12), and activation of on these genes was attributed to the loss of CaM sensitivity and not The Journal of Immunology 1185

to loss of sensitivity to an Id protein, because the losses of effect of anti-IgM were as complete with the m8N47 mutant that is only resistant to CaM as with the m847 mutant that is also resistant to Id proteins. This loss of effect of anti-IgM on the five genes was specific, because CaM-resistant m847 or m8N47 mutant of E12 did not affect the result of anti-IgM treatment for any control gene stimulated or inhibited by BCR activation, including IRF-4 (Fig. 5A) (12) (data not shown). To examine further whether the Ca2ϩ signaling from the BCR affected expression of Pax5, Bcl-6, MITF, Blimp1, Ets-1, and Fli-1 through CaM-mediated inhibition of E2A, we performed FACS analyses of primary splenic B cell cultures infected with retrovirus expressing wild-type or CaM-resistant E12 (Fig. 5, B and C, and representative FACS plots in supplemental Fig. S4).4 We found a clear difference between primary splenic B cells infected with wild-type E12 that showed Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Blimp1 expressions sensitive to anti-IgM treatment and cells infected with CaM-resistant m847 or m8N47 mutant of E12 that showed clearly anti-IgM-resistant expression of these genes (Fig. 5, B and C). The decrease in Bcl-6 expression upon BCR activation after infection with retrovirus expressing wild-type E12 was larger in cells activated with CD40L and IL-4 than with LPS and IL-4 (Fig. 5, cf B and C), a finding that agrees with the results from noninfected or vector-infected cells (Fig. 4, C and D). This larger decrease is probably due to the much higher initial activation of Bcl-6. In summary, the inhibitions of Pax5, Bcl-6, MITF, Ets-1, and Fli-1 expression and the activation of Blimp1 expression after BCR activation all depend on CaM sensitivity of E2A.

Plasma cell differentiation is regulated by CaM inhibition of E2A To investigate whether plasma cell differentiation is regulated by CaM and dependent on CaM sensitivity of E2A, we used the in vitro system for plasma cell differentiation (Fig. 1) and infected the purified splenic B cells with retrovirus expressing either CaM, wild-type E12, or CaM-resistant E12, followed by staining for the plasma cell marker syndecan-1 and gating for GFP-positive cells (infected cells) in the FACS analysis. Two days of anti-IgM stimulation of the BCR of B cells activated with LPS and IL-4 increased the frequency of large highly syndecan-1-expressing cells in the vector control infection (Fig. 6A), similarly to the results illustrated in Fig. 1. The generally higher frequencies of large syndecan-1-positive cells in Fig. 6 occur because gating for infected cells enriches for large highly syndecan-1-expressing cells, pre- sumably as a combined effect of more efficient infection of cells prone to become larger and more syndecan-1 expressing together with cells becoming more active and thereby larger due to the infection. Nevertheless, the effect of BCR stimulation on the frequency of large highly syndecan-1-expressing cells was even larger in Fig. 6A than in Fig. 1 (14.2% (33.7-19.5%) vs 6.4% (7.5-1.1%)). Importantly, overexpression of CaM resulted in an increase in large highly syndecan-1-expressing cells even without anti-IgM treatment. This increase, approximately as large as upon stimulation of the BCR (Fig. 6A), shows that overexpression of CaM has a corresponding positive effect on plasma cell differentiation as BCR activation. This result agrees with our findings on FIGURE 5. Loss of anti-IgM sensitivity of Pax5, Bcl-6, Ets-1, Fli-1, MITF, and Blimp1 expression by expression of CaM-resistant E12. A, E2A expression was reduced in DG75 B cells by a shuttle vector that expresses B and C, Average expression levels of the indicated proteins in cells in- shRNA targeting both E12- and E47-splice forms of human E2A mRNA. fected by retrovirus encoding wild-type E12 or CaM-resistant m847 or Shown are the effects of the shuttle vector that expresses shRNA interfering m8N47 mutant. B, The B lymphocytes from mouse spleen were activated with human E2A mRNA and complementation by expression of wild-type with LPS plus IL-4 and in C with CD40L plus IL-4. Expression levels of or mutant mouse E12 on the expression of the indicated mRNA. Where infected cells treated with anti-IgM for 3 h (MITF), 5 h (Pax5, Bcl-6, Ets-1, indicated, cells were treated with anti-IgM for 3 h before harvest. The and Fli-1), or 12 h (Blimp1) are expressed as percentage of the levels of the expression levels in cells transfected with empty pMEP4 vector and not corresponding infected cells not treated with anti-IgM. Results are mean treated with anti-IgM were set at 100%. Results are mean Ϯ SD (n ϭ 3). immunostaining fluorescence intensity Ϯ SD (n Ն 3). 1186 INITIATION OF DIFFERENTIATION TO PLASMA CELLS

factor whose expression is changed first (1–3, 17, 20). In this study, we show that the key signal in initiation of the plasma cell response, activation of the Ag receptor, does not lead to a key initial change in expression of one transcription factor, followed by a chain of secondary events, but instead leads to parallel changes in expression of several factors. The primary regulatory events identified were inhibition of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression. These inhibitions were very rapid; much of the reduction in their mRNA levels occurred within 30 min. The result was essen- tially the same after TLR activation with LPS and after stimulation FIGURE 6. CaM overexpression increases plasma cell differentiation, with the main Th cell cytokine CD40L. Thus, initiation of Ag-driven and CaM-resistant E12 reduces the differentiation. B lymphocytes from ASC development appears to occur in the same way both in T cell- mouse spleen were in A activated with LPS (2.5 ␮g/ml) plus IL-4 (1.25 independent and T cell-dependent differentiation. ng/ml) and in B activated with CD40L (50 ng/ml) plus IL-4 (1.25 ng/ml). Why are there many parallel changes rather than one key regulatory After 1 day, they were infected with retrovirus encoding CaM or the empty change? The former alternative has several advantages. It becomes MSCV vector, wild-type E12, or CaM-resistant m847 or m8N47 mutant, as indicated, for 1 day, followed by anti-IgM treatment, where more robust, because variation in expression or activity of one single indicated, for the 2 last days. Harvested cells were immunostained repressor of ASC differentiation will not alone have a decisive im- against syndecan-1 and analyzed by FACS. Cells highly expressing portance when several repressors together determine the critical de- GFP were considered infected and used to calculate the percentages of velopmental decision if the Ab of a particular B cell is good enough large cells (FSC-H Ն520) with increased syndecan-1 expression. Re- for ASC differentiation and thereby mass production of the Ab. Fur- sults are mean percentages Ϯ SD (n ϭ 4). thermore, the decision can be more fine-tuned by combining the strength of the BCR signal with the strengths of many other signals when several transcription factors, which are influenced to different the effects of overexpression of CaM on the regulation of Pax5, degrees by different signals, participate in the initial decision to dif- Bcl-6, MITF, Ets-1, Fli-1, and Blimp1 (Fig. 4, B–D). A corre- ferentiate or not to differentiate. In addition, BCR stimulation together sponding effect of overexpression of CaM on the frequency of with additional signals can also lead to differentiation into memory B large highly syndecan-1-expressing cells was obtained also in cells cells. The decision to differentiate into a memory B cell also depends grown in presence of CD40L and IL-4 (Fig. 6B). Cells grown with on transcription factor changes (2, 19), and parallel inhibition of ex- either LPS and IL-4 or CD40L and IL-4 and overexpressing wild- pression of several transcription factors would enable control of this type E12 were clearly affected by BCR activation, and a ϳ1.5- to decision between ASC or memory B cell differentiation by the level 2-fold higher frequency of large highly syndecan-1-expressing of the changes in different important transcription factors. A key dif- cells was observed, a frequency similar to the vector control-in- ference between these differentiations is that Pax5 is not down-regu- fected cells (Fig. 6). Importantly, in contrast to expression of the lated in memory B cell differentiation (2, 19). This difference could be wild type, expression of a CaM-resistant E12, either the Id-resis- the reason for the observed limitation of the Pax5 reduction (4- to tant m847 mutant or m8N47 that is sensitive to the Id proteins, 5-fold) upon the BCR stimulation, because a small fraction of the resulted in a reduced plasma cell differentiation both with and BCR-stimulated cells might differentiate into memory cells instead of without BCR stimulation, and this reduction appeared to be plasma cells. slightly larger upon BCR stimulation (Fig. 6). In summary, over- We found that the inhibitions of Pax5, Bcl-6, MITF, Ets-1, Fli-1, expression of CaM stimulates plasma cell differentiation, and the and Spi-B gene expression all depend on Ca2ϩ signaling, because efficiency of initiation of Ag receptor-driven plasma cell differen- they were blocked by Ca2ϩ chelator or a combination of Ca2ϩ chan- tiation depends on CaM sensitivity of E2A. nel blockers, and expression of all analyzed genes was reduced by the Ca2ϩ sensor CaM. Furthermore, the inhibitions of expression by BCR Discussion activation depended on CaM sensitivity of E2A when analyzed at the Plasmacytic differentiation is induced in response to appropriate mRNA level and/or at the protein level. The resistance of the expres- signals to generate specific Abs upon Ag exposure. The BCR plays sion of these genes to BCR activation when E2A is mutated does not a key role, and the strength of the interaction with Ag is critical in exclude that the other E proteins, E2-2 and HEB, may also contribute determining which clones enter the plasma cell response (4–6). to the expression and become CaM inhibited like E2A when the BCR Additional signals participating in plasma cell differentiation is activated. However, the reduction in mRNA for the analyzed pro- include pathogen-associated molecular patterns, which signal teins by shRNA against E2A in the DG75 cells and the dominant through TLRs, and cytokines and other T cell signals in which effect of CaM-resistant E12 over all endogenous CaM-sensitive E CD40L is of special importance (2, 17, 28). A large number of proteins suggest that E2A is the dominant E protein regulating these studies have identified several transcription factors that have to be genes. Thus, Ca2ϩ signaling leading to inhibition of E2A by Ca2ϩ- up-regulated or down-regulated in ASC differentiation. The differ- loaded CaM is needed for many gene expression changes in initiation entiation has been seen as a chain of changes in expression of of plasma cell differentiation. transcription factors (1–3, 17, 20), but it has been unclear which The very rapid and parallel reductions in the mRNAs for Pax5, regulatory change is primary and leads to subsequent changes in Bcl-6, MITF, Ets-1, Fli-1, and Spi-B by BCR stimulation, before expression of a series of other transcription factors. Reviews of this reduction of the protein levels, strongly suggest that these mRNA differentiation have argued for one or the other as the primary reductions are not secondary to another change in expression. E2A regulatory change (1–3, 17, 20). The up-regulation of Blimp1 has functions in hematopoietic progenitor cells to activate expression recently been excluded as the primary event, because initiation of of EBF and Pax5 and establishment of the program of B cell- the differentiation is Blimp1 independent, and up-regulation of specific gene expression (26, 31). The genes shown to be down- Blimp1 is very slow and secondary to down-regulations of repres- regulated through inhibition of E2A by Ca2ϩ-loaded CaM could sors during this differentiation (28–30). For a long time, Bcl-6 and all be direct targets of E2A. However, it is presently not known Pax5 have been key candidates for being the primary transcription whether they have an important E2A binding site in a regulatory DNA The Journal of Immunology 1187

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