Regulation of B cell differentiation by the ubiquitin-binding protein TAX1BP1

Tax1-binding protein 1 (TAX1BP1) is a ubiquitin-binding protein that restricts nuclear factor-κB (NF-κB) activation and facilitates the termination of aberrant inflammation. However, its roles in B-cell activation and differentiation are poorly understood. To evaluate the function of TAX1BP1 in B cells, we established TAX1BP1-deficient DT40 B cells that are hyper-responsive to CD40-induced extracellular signal-regulated kinase (ERK) activation signaling, exhibit prolonged and exaggerated ERK phosphorylation and show enhanced B lymphocyte-induced maturation protein 1 (Blimp-1; a transcription factor inducing plasma cell differentiation) expression that is ERK-dependent. Furthermore, TAX1BP1-deficient cells exhibit significantly decreased surface IgM expression and increased IgM secretion. Moreover, TAX1BP1-deficient mice display reduced germinal center formation and antigen-specific antibody production. These findings show that TAX1BP1 restricts ERK activation and Blimp-1 expression and regulates germinal center formation.

Scientific RepoRts | 6:31266 | DOI: 10.1038/srep31266 Tax1-binding protein 1 (TAX1BP1) was initially identified as a human T-cell leukemia virus type 1 Tax-binding protein 13 . TAX1BP1 functions as a ubiquitin-binding adaptor protein for the TNFα -inducible gene 3 (Tnfaip3)-encoded ubiquitin-modifying enzyme A20, which is composed of deubiquitinase and E3 ligase domains and inactivates K63-linked polyubiquitinated receptor-interacting protein kinase 1 (RIP1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) 14,15 . The complex formed by A20 and its regulatory molecule TAX1BP1 acts as a central negative regulator in multiple NF-κB-activating signaling pathways by cleaving K63-linked polyubiquitin chains and conjugating K48-linked polyubiquitin chains to its substrate, thereby inducing protein degradation 16 . In mice, targeting of TAX1BP1 causes hyperinflammations including inflammatory cardiac valvulitis and skin dermatitis through NF-κB dysregulation 15,17 . Cultured TAX1BP1-deficient cells are more hypersensitive to TNFα and IL-1β and exhibit increased NF-κB activation compared with wild-type (WT) cells. A20-deficient (Tnfaip3 −/− ) mice exhibit severe inflammation and cachexia and die prematurely. A20-deficient cells are hypersensitive to both lipopolysaccharide and TNFα stimulus and fail to terminate NF-κB responses. To regulate B-cell responses, A20 restricts CD40-induced NF-κB signals that repress Fas-mediated cell death, and B-cell-specific A20-deficient mice display elevated germinal center B-cell numbers and autoantibody production 18,19 . These data suggest that TAX1BP1 may play a role in B-cell differentiation; however, the role of TAX1BP1 in regulating B-cell responses remains unknown.
In this study, we generated TAX1BP1-deficient DT40 B cell lines to address the role of TAX1BP1 in B cells. The chicken B-cell line DT40 expresses surface IgM, but it continues Ig gene conversion with apparent arrest at the bursal B cell stage 20 . TAX1BP1-deficient DT40 cells exhibited a plasmacytic phenotype with impaired cell surface IgM expression, significantly enhanced ERK phosphorylation, and increased expression of the plasma cell transcription factors Blimp1, IRF4, and XBP1. In mice, targeting of TAX1BP1 led to impaired GC B cells and GC formation and a subsequent decrease in antigen-specific antibody production. These results demonstrate that TAX1BP1 is required for B-cell differentiation and GC formation.

Results
Targeted disruption of TAX1BP1 in DT40 cells. To identify the role of TAX1BP1 in B cells, we generated TAX1BP1 knockout cells in the chicken B-cell line DT40. The chicken Tax1bp1 gene is located on chromosome 2, which is trisomic in DT40 cells. We generated deletion constructs comprising different marker genes (bsr, his, and ecogpt), integration of which into the first exon of the Tax1bp1 allele resulted in a deletion of the coding base pairs 1-179 as previously described 21 (Fig. 1a). Tax1bp1 gene disruption was verified by Southern blot analysis using the indicated 5′ probe (Fig. 1b). Reverse transcription PCR analysis confirmed that TAX1BP1 −/−/− DT40 cells did not express TAX1BP1 transcripts (Fig. 1c). In addition, we confirmed the expression of TAX1BP1 protein in WT DT40 cells but not in TAX1BP −/−/− cells using a specific TAX1BP1 antibody (Fig. 1d). To examine the functional effects of TAX1BP1 on NF-κB activation in B cells, we measured transcriptional activity using an NF-κB-responsive reporter. Disruption of TAX1BP1 enhanced both LPS and anti-CD40 antibody (αCD40)-mediated NF-κB activation compared with WT DT40 cells (Fig. 1e). (d) Immunoblot with TAX1BP1 antibody. (e) TAX1BP1 inhibits LPS-and anti-CD40 antibody (αCD40)induced NF-κB activation in B cells. TAX1BP1-deficient DT40 cells (BP1 −/−/− ) exhibited higher NF-κB activity in response to lipopolysaccharide (LPS) and αCD40 compared with WT cells. Cells were transfected with an NF-κB-luc reporter and a renilla reporter pRL-CMV plasmid as an internal control. At 24 h after transfection, the indicated concentrations of αCD40 (2.5 μg/ml) and LPS (50 ng/ml) were added for 8-24 h, after which the cells were harvested and subjected to a luciferase assay. Relative NF-κB activation is shown in comparison to the activation level in unstimulated WT cells (arbitrarily set at 1). The results are shown as the means and standard errors of the mean (s.e.m.; n = 3). *P < 0.05, P values are based on two-tailed Student's t tests.
Scientific RepoRts | 6:31266 | DOI: 10.1038/srep31266 TAX1BP1 restricts ERK activation in B cells. B cells are regulated by several stimuli through various receptors such as BCR, TLR, and CD40. Signaling through CD40, a TNF receptor family member, induces NF-κB activation, resulting in B cell activation and survival 22 . TAX1BP1 has been shown to interact with A20 to form a ubiquitin-editing complex that functions as a negative regulator for NF-κB activation via TRAF6 and RIP1 degradation 14,23 . A20-deficient B cells display enhanced CD40-induced canonical NF-κB activation, evidenced by increased inhibitor of kappa B (I-κB) phosphorylation and degradation, but not MAPK pathway activation 18 . To examine whether TAX1BP1 similarly restricts CD40-induced NF-κB signals, we stimulated WT and TAX1BP1 −/−/− DT40 cells with αCD40 for the indicated time intervals. TAX1BP1-deficient B cells exhibited slightly enhanced I-κB phosphorylation (Fig. 2a). Next we examined the role of TAX1BP1 in the activation of MAPKs, including JNK and ERK. We detected significantly elevated and prolonged ERK phosphorylation in TAX1BP1 −/−/− DT40 cells. The ERK phosphorylation level was calculated as the ratio of phospho-ERK to total ERK protein and was normalized with respect to unstimulated WT cells (Fig. 2a). In addition, the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 reduced ERK phosphorylation in TAX1BP1-deficient cells (Fig. 2b). Taken together, TAX1BP1 deficiency in DT40 cells selectively enhances activation of the ERK pathway in response to αCD40. To confirm the physiological role of TAX1BP1 in mouse B lymphocyte activation, we isolated splenic B cells from TAX1BP1-deficient (TAX1BP1 −/− ) 15 and WT mice and measured activation of the NF-κB and ERK pathway. Figure 2c shows that ERK phosphorylation was significantly enhanced in αCD40-stimulated cells, and the MEK inhibitor U0126 reduced ERK phosphorylation in B cells from TAX1BP1 −/− mice (Fig. 2d). Previous studies have shown that TRAF6 contributes to the CD40-mediated activation of ERK and cell proliferation in lymphoid cells and tumor cells 24,25 . Following CD40 activation, the K63-specific ubiquitin ligase activity of TRAF6 is rapidly stimulated, leading to TRAF6 autoubiquitination. These resulting polyubiquitin chains may further stabilize the respective signaling complexes and induce ERK activation. We therefore hypothesized that TAX1BP1 inhibits TRAF6 polyubiquitination in these signals and restricts ERK activity. As expected, upon αCD40 stimulation TRAF6 K63-linked polyubiquitination was enhanced in TAX1BP1-deficient cells compared to WT DT40 cells ( Supplementary Fig. S1). TAX1BP1 negatively regulates expression of the gene encoding Blimp-1 and B cell differentiation. We restored TAX1BP1 expression in TAX1BP1 −/−/− cells (TAX1BP1 −/−/− /Tax1bp1) to provide functional complementation and verified the disruption of TAX1BP1-enhanced ERK phosphorylation in B cells and restoration of TAX1BP1-mediated attenuation of ERK activity (Fig. 3a). The ERK signaling pathway is essential for the differentiation of B cells into antibody-secreting plasma cells because it induces the expression of Prdm1, which encodes the plasma cell master regulator Blimp-1 26 . According to quantitative PCR analysis, TAX1BP1 deficiency significantly enhanced the expression of Blimp1 compared with WT cells, and the restoration of TAX1BP1 expression restored Blimp-1 suppression (Fig. 3b). The MEK inhibitor U0126, previously shown to reduce ERK phosphorylation, also suppressed Blimp-1 expression in TAX1BP1-deficient cells (Fig. 3c). In addition, TAX1BP1-deficient cells exhibited reduced expression levels of Pax5 and Bcl-6, which are repressed by Blimp-1, whereas the expression of IRF4, which is induced by the NF-κB pathway, was significantly enhanced compared with WT cells (Fig. 3d). To examine the physiological role of TAX1BP1 in expression of Blimp1, we isolated splenic B cells from WT and TAX1BP1 −/− mice and measured Blimp1 expression. Even without CD40  S2a). Antibody-secreting plasma cells feature a constitutively active unfolded protein response (UPR) for which the transcription factor XBP1 is required. During plasma cell differentiation from stimulated B cells, active UPR signaling leads to the enzymatic splicing of XBP1 mRNA, thereby enabling expression of the transcription factor XBP1 and inducing cellular structural changes to facilitate high antibody production rates 27,28 . According to quantitative PCR analysis, total XBP1 mRNA expression was significantly increased in TAX1BP1 −/−/− cells compared with WT cells, whereas the reintroduction of TAX1BP1 attenuated this increase (Fig. 4b). In addition, we analyzed the expression levels of spliced and unspliced XBP1 mRNA. In TAX1BP1 −/−/− cells, we observed a significant increase in spliced XBP1 (XBP1sp) mRNA expression level compared with WT DT40 cells and TAX1BP1 −/−/− /Tax1bp1 cells (Fig. 4c). We next examined surface IgM expression. As expected, surface IgM expression was decreased in TAX1BP1 −/−/− cells compared with WT cells, and reintroduction of TAX1BP1 reconstituted surface IgM expression (Fig. 4d). We also confirmed the decreased surface IgM expression and induced XBP1 mRNA splicing (XBP1sp) in splenic B cells from TAX1BP1 −/− mice compared with WT mice (Supplemental Fig. S2b,c). These data suggest that TAX1BP1 disruption enhanced splicing of the membrane-type IgM heavy chain (μM) mRNA to the secretory type and enhanced the observed aberrant differentiation into antibody-secreting plasma cells. We further assessed the B cell development in  Splenic germinal center hypoplasia in TAX1BP1-deficient mice. To study the physiological role of TAX1BP1 in B-lymphocyte activation and differentiation and GC formation, we immunized WT and TAX1BP1 −/− mice intraperitoneally using sheep red blood cells (SRBCs), a potent T-cell-dependent antigen that induces GC formation. After the second immunization, we isolated splenic B cells from the immunized WT and TAX1BP1 −/− mice and measured the gene expression levels of transcription factors involved in GC formation. According to our quantitative PCR analysis, splenic B cells from SRBC-immunized TAX1BP1 −/− mice expressed much higher levels of Blimp-1 when compared with WT cells, similar to the previous findings in TAX1BP1-deficient DT40 cells (Fig. 5a). Xbp-1 expression, which is induced by Blimp-1, was also upregulated in TAX1BP1 −/− mice, whereas the expression of Pax5 and Bcl-6, which are repressed by Blimp-1, was downregulated in TAX1BP1-deficient splenic B cells (Fig. 5a).
Furthermore, we used flow cytometry to evaluate GC B cells in single-cell suspensions of splenic B cells isolated from SRBC-immunized WT and TAX1BP1 −/− mice. The GL7 monoclonal antibody reacts with a cell-surface protein found on activated GC B cells. Notably, the population of these splenic GC B cells coexpressing the GC markers GL7 and Fas 29,30 was reduced in SRBC-immunized TAX1BP1 −/− mice compared with WT mice (Fig. 5b). We also used flow cytometry to compare the frequency of plasma cells and plasmablast cells, which express cell surface antigen CD138 (syndecan-1) and low levels of B220 31,32 , among splenic B cells isolated from WT and TAX1BP1 −/− mice after SRBC immunization. This analysis revealed that TAX1BP1 −/− mice exhibited profound reduction of plasma cells compared with WT mice after SRBC immunization (Fig. 5c).
This finding was further confirmed by a histological analysis of hematoxylin-eosin (H&E) stained splenic sections. Prominent GC formation was observed in the spleens of SRBC-immunized WT mice; in contrast, GC formation was impaired in the spleens of SRBC-immunized TAX1BP1 −/− mice (Fig. 6a). Examination of spleens by immunohistochemistry also showed that TAX1BP1 −/− mice displayed significant reductions in GC size and number compared with WT mice at day 10 after SRBC immunization (Fig. 6b-d). IgM + plasma cells were similarly present in spleen sections of WT and TAX1BP1 −/− mice, however, IgG + plasma cells were reduced in TAX1BP1 −/− mice (Fig. 6b). We next sought to determine whether the reduced GC formation and plasma cell differentiation in TAX1BP1 −/− mice would affect antigen-specific antibody production. Accordingly, we evaluated the serum levels of SRBC-specific IgM and IgG1 antibodies from WT and TAX1BP1 −/− mice following SRBC immunization. Both WT and TAX1BP1 −/− mice exhibited elevated serum SRBC-specific IgM (Fig. 6e) and IgG1 titers. However, TAX1BP1 −/− mice had lower SRBC-specific IgG1 titers compared with WT mice. These findings suggest that TAX1BP1 regulates both GC formation and specific antibody production.

Discussion
On encountering antigen, B cells alter their physiological state and localization and initiate differentiation through a GC response. GCs are critical for the generation of memory B cells and plasma B cells that produce high-affinity antibodies; a process strictly regulated by specific transcription factors such as Bcl-6 and Blimp-1 2,33 .
Bcl-6-deficient mice exhibit defective T-cell-dependent immune responses as a result of completely abolished GC formation, whereas constitutive Bcl-6 expression inhibits B cell plasma cell differentiation 34,35 . B cells must switch off Bcl-6 expression and switch on transcription factors such as Blimp-1 to exit the GC and differentiate into plasma cells. Blimp-1 is required for plasma cell differentiation because it represses a large set of genes required for GC B-cell development and cell proliferation (e.g., Bcl-6) and executes a plasma cell-specific transcription program established by IRF4, Blimp-1, and X-box-binding protein 1 (XBP1) 8,36,37 . In response to NF-κB signaling, IRF4 expression directly activates Blimp-1 to promote plasma cell differentiation 38,39 and counteract Bcl-6-mediated repression of the Blimp-1 promoter 40 . In addition, ERK activation is critical for inducing the expression of Blimp-1 26 . Recently, constitutive Blimp-1 activation was found to impair the differentiation of GC B cells in mice lacking Srg3, a component of the SWI/SNF complex that acts with Bcl-6 to repress the Blimp-1 promoter 41 . Taken together, these findings indicate that the regulation of Blimp-1 expression is critical for GC formation and plasma cell differentiation; however, the involvement of these intracellular signaling systems in Blimp-1 repression remains unknown. TAX1BP1, a ubiquitin binding protein, functions as an adaptor to recruit its catalytic partner A20 to K-63 polyubiquitinated proteins such as TRAF6 and RIP1. This ubiquitin-editing enzyme combines with Itch and RNF11 to negatively regulate NF-κB signaling pathways 15,23 . TNF or interleukin 1 (IL-1)-induced TAX1BP1 phosphorylation by IKKα is pivotal to the assembly of this quadruple protein complex and termination of NF-κB signaling 42 . A20-deficient B cells exhibit enhanced NF-κB pathway activation in response to αIgM, LPS, and αCD40 stimulation; however, MAPK pathway activation and AKT phosphorylation remain unchanged 18,43 . Mice with B-cell-specific reduction of A20 expression harbor elevated numbers of GC B cells, autoantibodies, and glomerular immunoglobulin deposits 43 , and A20-deficient B cells are resistant to Fas-mediated cell death, likely because of the increased expression of NF-κB-dependent antiapoptotic proteins such as Bcl-x. These findings indicate that A20 restricts B cell survival, but it protects other cells from TNF-induced cell death. Despite studies demonstrating that A20 restricts autoimmune responses, it has remained unclear how A20 and its partner TAX1BP1 regulate B cells or plasma cell transcription factors.
To investigate the role of TAX1BP1 in B-cell activation and plasma cell differentiation, we generated a TAX1BP1-deficient line of DT40 B cells. The depletion of TAX1BP1 in DT40 cells caused a slight increase in NF-κB pathway activation in response to B cell activation via LPS and αCD40. TAX1BP1-deficient DT40 cells also showed an increased level of TRAF6 polyubiquitination, which contributes to CD40-mediated ERK activation 24,44 (Supplementary Fig. S1). Thereby, ERK phosphorylation was significantly increased and prolonged in TAX1BP1-deficient DT40 cells and splenic B cells from TAX1BP1 −/− mice (Fig. 2) in contrast to A20-deficient B cells in which Erk phosphorylation was not altered 18 . TAX1BP1 restoration attenuated the constitutive ERK signaling observed in TAX1BP1-deficient cells (Fig. 3). TAX1BP1 may restrict CD40-induced ERK phosphorylation in an A20-independent pathway. It will be interesting to investigate whether A20 and TAX1BP1 signaling pathways can act independently to regulate ERK activity in GC formation and B-cell differentiation. In B cells, ERK pathway activation is critical for inducing Blimp-1 26 . TAX1BP1 disruption enhanced ERK activity and induced constitutively higher expression levels of Blimp-1, and treatment with the MEK inhibitor U0126 inhibited the expression of Blimp-1 (Fig. 3). Xbp1, which is induced by Blimp-1, was also strongly expressed, whereas the expression levels of Pax5 and Bcl-6, which are repressed by Blimp-1, were significantly reduced. We also confirmed that constitutive Blimp-1 expression in TAX1BP1-deficient DT40 B cells leads to spontaneous plasmacytic differentiation (Figs 3 and 4). TAX1BP1 may regulate the termination of αCD40-induced NF-κB and ERK signaling, thereby restricting the continuous expression of Blimp-1.
CD40 induction is a critical step in the T-cell-dependent immune response 45,46 that contributes to GC formation, memory B-cell development, Ig isotype switching, and affinity maturation. Previous in vivo experiments have demonstrated that constitutively active CD40 receptors can prolong B-cell survival and increase proliferation; however, these B cells exhibit impaired GC formation 47 . To evaluate the physiological role of TAX1BP1 in the adaptive immune response, we immunized WT and TAX1BP1 −/− mice intraperitoneally with SRBCs. The splenic B cells from SRBC-immunizes TAX1BP1 −/− mice showed high expression of Blimp-1 and downregulation of Bcl-6, a critical regulator of the GC reaction, similar to TAX1BP1-deficient DT40 cells (Fig. 5a). Despite the induction of a strong GC reaction in WT mice, TAX1BP1 −/− mice exhibited impaired GC formation, resulting in the attenuation of plasma cell and antigen-specific antibody production (Figs 5 and 6). These results suggest that Blimp-1 activation induced aberrant plasma cell differentiation and downregulation of Bcl-6 expression impaired the germinal center formation and then leading significant reduction of plasma cells in TAX1BP1 −/− mice compared with WT mice after SRBC immunization (Fig. 5c).
Our results demonstrate that in B cells, TAX1BP1 restricts CD40-mediated responses and terminates NF-κB and ERK signaling to restrict aberrant Blimp-1 expression, regulate plasma cell development, and initiate GC formation. Accordingly, TAX1BP1 appears to be a key regulator of GC reactions.

Generation of TAX1BP1 deficient DT40 cells (Bsr/Ecogpt/His).
The Tax1bp1 gene was disrupted by gene targeting. The targeting constructs were designed to replace the first exon with the HisD, Bsr or Ecogpt resistance gene cassette. The gene disruption is expected to remove the genomic region that encodes chicken TAX1BP1 amino acids 1 to 81. The linearized targeting vector was introduced into WT DT40 cells. Transfections by electroporation and selections of the clones were done as previously described 48 . The disruption of Tax1bp1 gene was screened by genomic southern blotting using probe indicated in Fig. 1 and genome DNA digested by HindIII. The TAX1BP1 probe was amplified by PCR using chicken genomic DNA as a template with the following primers: (forward: 5′-CATGATCCTGTGATTCGATGACGTGC-3′, reverse: 5′-GATGCTACTTTATGGAATACGCTGGGAG-3′). All the methods involved in DNA recombination were approved by Committees of Tokyo University of Pharmacy and Life Sciences (Tokyo, Japan) and carried out in accordance with approved University guidelines. Animal Care and Committees of Tokyo University of Pharmacy and Life Sciences (Tokyo, Japan). All mice were maintained under specific pathogen-free conditions. All the methods were carried out in accordance with the Guidelines for Animal Experiments of Tokyo University of Pharmacy and Life Sciences (Tokyo, Japan).
Luciferase assays. Cells were transfected with an NF-κB luciferase reporter (Clontech) and the Renilla reporter pRL-CMV (Promega) as an internal control. Cells were harvested 24 h after transfection and incubated with or without indicated reagents, 2.5 μg/ml anti-CD40 antibody (αCD40) (AbD Serotec) and 50 ng/ml LPS (Sigma) for 8-24 h, and cell lysates were analyzed by dual-luciferase assay according to the manufacturer's instructions (Promega). Results presented are the average of triplicate experiments with the S.D. values shown as error bars.
Co-immunoprecipitation and immunoblotting. WT DT40 cells and TAX1BP1 −/−/− cells were incubated for indicated time after CD40 activation and lysed in RIPA buffer. Lysates were incubated with anti-TRAF6 antibody (abcam) or relative non-immunized IgG for 3 h at 4 °C followed by protein G Sepharose for another 2 h. Proteins were detected by anti-TRAF6 (Santa Cruz Biotechnology), anti-β-actin (SIGMA) and anti-K63 Ubiquitin (Millipore).
Enzyme-linked immunosorbent assay (ELISA). Immunoglobulin concentrations in the serum and cell medium were measured by standard sandwich ELISA. Concentrations of antibodies specific for SRBCs were determined by ELISA using the mouse anti-SRBC ELISA kit (Life Diagnostics, Inc.), according to the manufacturer's instructions. Concentrations of chicken IgM were determined by ELISA using chicken IgM ElISA kit (Bethyl Laboratories, Inc), according to the manufacturer's instructions.