Survival of Anergic B Cells B Activation and Κ BAFF-Regulated NF

Critical Role of B Cell Lymphoma 10 in BAFF-Regulated NF- κB Activation and Survival of Anergic B Cells

This information is current as Mei Yu, Yuhong Chen, Yinghong He, Andrew Podd, of September 30, 2021. Guoping Fu, Jacqueline A. Wright, Eden Kleiman, Wasif N. Khan, Renren Wen and Demin Wang J Immunol 2012; 189:5185-5193; Prepublished online 19 October 2012;

doi: 10.4049/jimmunol.1102952 Downloaded from http://www.jimmunol.org/content/189/11/5185

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Critical Role of B Cell Lymphoma 10 in BAFF-Regulated NF-kB Activation and Survival of Anergic B Cells

Mei Yu,*,† Yuhong Chen,† Yinghong He,*,† Andrew Podd,†,‡ Guoping Fu,† Jacqueline A. Wright,x Eden Kleiman,x Wasif N. Khan,x Renren Wen,† and Demin Wang*,†,‡

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-kB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimu- Downloaded from lation induced Bcl10 association with IkB kinase b, a key component of the canonical NF-kB pathway. Consistently, Bcl10- deficient B cells were impaired in BAFF-induced IkBa phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10- deficient B cells also displayed reduced expression of NF-kB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-xL, a BAFF-induced NF-kB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-kB activation directly and noncanonical NF-kB activation indirectly. The BAFF-R/Bcl10/NF-kB signaling axis plays a critical role in peripheral B cell tolerance http://www.jimmunol.org/ by regulating the survival of self-reactive anergic B cells. The Journal of Immunology, 2012, 189: 5185–5193.

ewly produced B cells, 50–75% of which are estimated to B cells become anergic (11, 18). As much as 5–10% of peripheral be self-reactive B cells (1, 2), undergo negative selection B cells are anergic, and given the fast turnover rate of these cells, N by three distinct mechanisms in the bone marrow: re- anergy likely represents a major physiological mechanism for ceptor editing, clonal deletion, and anergy (3–8). The signaling establishing immune tolerance (19). In the periphery, although BCR threshold of a self-reactive BCR has long been thought to deter- signaling is required for the maintenance of normal and self-reactive mine the negative selection mechanism. A highly self-reactive anergic B cells (20, 21), TNF family member BAFF is essential for by guest on September 30, 2021 BCR initiates new rearrangements of V genes to change recep- the survival of these B cells (22–24). Importantly, self-reactive tor specificity by receptor editing (9–13), or it drives B cells to anergic B cells have increased dependence on BAFF for survival undergo apoptosis and be eliminated by clonal deletion (14, 15). A compared with normal B cells (24, 25). Excess BAFF mainly moderately self-reactive BCR drives B cells to become unre- rescues self-reactive anergic B cells from competitive elimination sponsive to Ags, a state termed anergy (16, 17). After emerging (24, 25). Thus, in addition to BCR, BAFF plays a crucial role in from the bone marrow, transitional B cells undergo further mat- sustaining the survival of anergic B cells in the periphery. uration and selection in the periphery. Highly self-reactive B cells BAFF supports the survival of B cells through its cognate re- are removed by clonal deletion, whereas moderately self-reactive ceptor. Although BAFF binds to three receptors, that is, BAFF-R, B cell maturation Ag, and TACI, BAFF-R plays the predominant role in BAFF function (22, 23). Deficiency of BAFF-R impairs *State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nan- jing 210093, People’s Republic of China; †Blood Research Institute, BloodCenter of the survival of peripheral B cells similar to deficiency of BAFF, Wisconsin, Milwaukee, WI 53226; ‡Department of Microbiology and Molecular x resulting in nearly complete loss of peripheral B cells (22, 26, 27). Genetics, Medical College of Wisconsin, Milwaukee, WI 53226; and Department In contrast, deficiency of B cell maturation Ag impairs the survival of Microbiology and Immunology, University of Miami, Miami, FL 33136 of long-lived plasma cells (28), whereas lack of TACI leads to a 2- Received for publication October 12, 2011. Accepted for publication September 27, 2012. to 3-fold increase in mature B cell number (29). This work was supported in part by National Institutes of Health Grants R01 One important signaling event of BAFF-R that leads to B cell AI079087 (to D.W.), P01 HL44612 (to D.W.), and R21 AI088511 (to W.N.K.); by survival is the NF-kB–dependent upregulation of Bcl-2 family a Scholar Award from the Leukemia and Lymphoma Society (to D.W.); by a grant prosurvival members (22, 30). NF-kB/Rel family of transcription from the Advancing a Healthier Wisconsin endowment fund (to D.W.); and by American Recovery and Reinvestment Act Supplement R01 AI060729-06S1 (to factors consists of five members, that is, RelA/p65, c-Rel, RelB, W.N.K. to support J.A.W.). NF-kB1 (p105/p50), and NF-kB2 (p100/p52), which can form Address correspondence and reprint requests to Dr. Demin Wang, Blood Research homo- and heterodimers (31, 32). NF-kB dimers exist as inactive Institute, BloodCenter of Wisconsin, 8727 Watertown Plank Road, Milwaukee, WI complexes bound to the inhibitor of NF-kB(IkB), such as IkBa 53226. E-mail address: [email protected] or IkBb, in resting cells (33). There are two major signaling path- Abbreviations used in this article: Bcl10, B cell lymphoma 10; CARMA1, caspase recruitment domain membrane-associated guanylate kinase protein 1; CBM, ways that mediate the activation of NF-kB, known as the canonical CARMA1/Bcl10/MALT1; FO, follicular; HEL, hen egg lysozyme; IKK, IkB kinase; and noncanonical pathways (34). It is known that BAFF-R is able to MALT1, MALT lymphoma translocation protein 1; PKC, protein kinase C; PLC, induce NF-kB activation through both pathways (35–40). phospholipase C; sHEL, soluble hen egg lysozyme. Upon BAFF binding, BAFF-R induces a cellular inhibitor Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 of apoptosis 1/2 and TNFR-associated factor 2 ubiquitin ligase www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102952 5186 Bcl10 IN BAFF-INDUCED NF-kB ACTIVATION complex-dependent degradation of TNFR-associated factor 3, B cell isolation preventing NF-kB–inducing kinase degradation and resulting in The splenic CD93+ immature (also known as AA4.1+)andCD932 (AA4.12) the subsequent activation of the noncanonical NF-kBpathway mature B cells were isolated from wild-type and Bcl10-deficient mice as (37, 38). NF-kB–inducing kinase activates IkBkinase(IKK)a, previously described (52). Briefly, splenic B cells were isolated by negative andactivatedIKKa phosphorylates p100, leading to processing selection using anti–CD4-, anti–CD8-, and anti–CD11b-coated MACS of NF-kB2 from a p100 precursor to a p52 product. Then p52 microbeads (Miltenyi Biotec) and then stained with biotin-conjugated anti- CD93 (eBioscience). Immature B cells (CD93+) were isolated using dimerizes with RelB to form the p52/RelB active heterodimer streptavidin-conjugated microbeads with the flow through constituting the that translocates to the nucleus and regulates gene expression mature B cells (CD932). For NF-kB2/p100 mRNA expression studies, (35, 36). Additionally, BAFF-R can activate NF-kB through the mature B cells were also isolated from CD93-depleted splenocytes by canonical pathway that involves the activation of the IKK positive selection using biotin-conjugated anti-CD23 (eBioscience) Abs and streptavidin-conjugated microbeads. Purity of CD93+ and CD932 complex consisting of IKKa, IKKb, and NEMO (39, 40). Acti- CD23+ B cells was ∼90 and 95%, respectively, as determined by anti-B220 vated IKK complex induces the phosphorylation and subsequent Ab staining followed by flow cytometry. degradation of IkBs. Removal of IkBs results in the nuclear translocation of p50-containing NF-kB heterodimers and subse- Immunoprecipitation and Western blotting analysis quent initiation of gene transcription. Despite progress in this area Splenic AA4.12 mature B cells (1 3 106) from wild-type and Bcl10- of research, the mechanisms by which BAFF-R mediates the ac- deficient mice were stimulated with BAFF (100 ng/ml) at 37˚C for the tivation of NF-kB in both the canonical and noncanonical path- indicated times and then lysed in lysis buffer (10 mM Tris-HCl [pH 7.5], m m ways are not fully understood. 150 mM NaCl, 1% Nonidet P-40, 3 g/ml aprotinin, 2 g/ml pepstatin, 2 mg/ml leupeptin). Cell lysates were subjected to Western blotting anal- B cell lymphoma 10 (Bcl10) is an adaptor protein characterized by ysis with the indicated Abs. For detecting IkB phosphorylation, splenic Downloaded from an N-terminal caspase recruitment domain and a C-terminal Ser/Thr- mature B cells were pretreated with MG132 for 30 min before BAFF 2 rich region (41–45). Bcl10 is essential for BCR-mediated NF-kB stimulation. For coimmunoprecipitation, wild-type AA4.1 mature B cells 3 7 activation (46, 47). Bcl10, along with caspase recruitment domain (2 10 ) were stimulated with BAFF (200 ng/ml) or anti-IgM (10 mg/ml) for the indicated times. Cell lysates were precleared with Sepharose beads membrane-associated guanylate kinase protein 1 (CARMA1) and and subsequently incubated with anti-Bcl10 Ab-conjugated Sepharose MALT lymphoma translocation protein 1 (MALT1), forms a three- beads (sc-5273 AC; Santa Cruz Biotechnology) at 4˚C overnight. After component complex that couples BCR-induced protein kinase C washing five times with cold lysis buffer, bound proteins were eluted in http://www.jimmunol.org/ (PKC) activity to IKK complex activation (48–51). Bcl10 defi- 23 Laemmli buffer at 95˚C for 5 min and subjected to 10% SDS-PAGE, ciency impairs BCR-induced NF-kB activation (47). Additionally, followed by Western blotting analysis with the indicated Abs. Rabbit polyclonal anti-ERK (sc-093), anti-p50 (sc-114), anti-p65 (sc-109), Bcl10 plays a critical role in TCR-, FcεR-, and TLR4-induced NF- anti–c-Rel (sc-071), and anti-RelB (sc-226), and mouse monoclonal anti-p52 kB activation, and its deficiency impairs NF-kB activation by (sc-7386), anti–phospho-ERK (pThr202/pTyr204, sc-7383), anti-Bcl10 (sc- these receptors (46). Although Bcl10 plays a critical role in NF- 5273), and anti-YY1 (sc-7341) were purchased from Santa Cruz Biotech- kB activation by multiple receptors, its role in BAFF-R–mediated nology. Mouse monoclonal anti-MALT1 was a generous gift from Dr. Vishva Dixit (Genentech, San Francisco, CA). Mouse monoclonal anti–phospho- NF-kB activation is not known. IkBa (Ser32/36, 9246), rabbit polyclonal anti–phospho-Akt (phospho-Ser473, Our current studies find that Bcl10 directly controls BAFF- 9271) and anti-IkBa (9242), and rabbit monoclonal anti-IKKb (2C8, 2370) mediated canonical NF-kB activation and induces the expression were purchased from Cell Signaling Technology. Rabbit polyclonal anti– by guest on September 30, 2021 of NF-kB2/p100, thus indirectly regulating BAFF-mediated non- Bcl-xL (B22630) was purchased from BD Transduction Laboratories. Hamster canonical NF-kB activation. This BAFF-R/Bcl10/NF-kB signaling monoclonal anti-Bcl2 (554218) was purchased from BD Biosciences. Mouse monoclonal anti-GADPH (MAB374), anti–Bcl-10 (AB16506), and anti-actin axis specifically supports the survival of self-reactive anergic B cells (MAB1501R) were purchased from Chemicon International/Millipore. in vivo. Impairment of this axis by Bcl10 deficiency alters the peripheral tolerance mechanism for self-reactive B cells, switching NF-kB gel mobility shift assay from anergy to deletion. Splenic CD932 mature B cells (1 3 106) from wild-type and Bcl10-deficient mice were stimulated with BAFF (200 ng/ml) for 3 or 16 h and then lysed in Materials and Methods the lysis buffer (20 mM HEPES pH 7.9, 350 mM NaCl, 1 mM MgCl2, Mice 0.5 mM EDTA, 0.5 mM DTT, 20% glycerol, 1% NP-40). Cell lysates were incubated with 32P-labeled NF-kB probe (59-AGTTGAGGGGACTTTCC- Bcl10-deficient mice have been previously described (47). Heterozygous CAGGC-39; Santa Cruz Biotechnology) for 15 min at room temperature, Bcl10-deficient mice were bred to generate wild-type control and Bcl10- HEL resolved on a 4% polyacrylamide gel at 4˚C, and exposed to x-ray film. For deficient mice. Ig hen egg lysozyme (Ig ) transgenic mice (C57BL/6 supershift, cell lysates were incubated with the indicated Abs for 15 min MD4) and soluble HEL (sHEL) transgenic mice (C57BL/6 ML5) were ob- before adding the probes. tained from The Jackson Laboratory. These transgenic mice were bred with 2 heterozygous Bcl10+/ mice to generate wild-type IgHEL, Bcl10-deficient Preparation of cytoplasmic and nuclear extracts IgHEL, wild-type IgHELsHEL, and Bcl10-deficient IgHELsHEL mice. 2 6 Mice used for the experiment were generally 2–4 mo old except where Splenic CD93 mature B cells (2 3 10 ) from wild-type and Bcl10-deficient specifically indicated. All mouse procedures were approved by the In- mice were stimulated with BAFF (100 ng/ml) for 16 h. The cells were stitutional Animal Care and Use Committee. collected and suspended in 100 ml cold buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.2 mM EDTA, 1 mM DTT, 3 mg/ml aprotinin, 2 mg/ml Flow cytometry pepstatin, and 1 mg/ml leupeptin). After incubation on ice for 15 min, Nonidet P-40 was added to a final concentration of 0.5%. The mixtures were Single-cell suspensions from the spleen of the experimental and control vortexed for 10 s and spun at 16,000 3 g for 30 s. The supernatants were mice were treated with Gey’s solution to lyse RBCs and resuspended in collected as the cytoplasmic extracts. The pellets were washed with buffer A PBS with 2% FBS. The cells were then stained with a combination of once and resuspended in 50 ml buffer B (20 mM HEPES [pH 7.9], 400 mM fluorescence-conjugated Abs. PE-Cy7–conjugated anti-CD19 (25-0193), NaCl, 2 mM EDTA, 1 mM DTT, 3 mg/ml aprotinin, 2 mg/ml pepstatin, and allophycocyanin-conjugated anti-IgM (17-5790), FITC-conjugated anti– 1 mg/ml leupeptin), followed by incubation on ice for 15 min. The mixtures BAFF-R (11-5943), Cy-Chrome–conjugated anti-B220 (15-0452), PE- were spun at 16,000 3 g for 5 min and the supernatants were collected as the conjugated anti-B220 (12-0452), biotin-conjugated anti-AA4.1 (CD93; nuclear extracts. 13-5892), PE-conjugated anti-CD23 (12-0232), and PE-Cy7–conjugated streptavidin (25-4317) were purchased from eBioscience. PE-conjugated Quantitative RT-PCR anti-IgD (1120-09) and FITC-conjugated anti-IgM (1140-02) were purchased from SouthernBiotech. Allophycocyanin-conjugated streptavidin Splenic CD932CD23+ mature or CD93+ immature B cells from wild-type (554067) was purchased from BD Pharmingen. Stained cells were ana- and Bcl10-deficient mice were stimulated with BAFF (100 ng/ml) for 4 or lyzed on a BD LSR II cytometer with BD FACSDiva software. 16 h. RNA was extracted from the cells using an RNeasy Mini kit (Qiagen) The Journal of Immunology 5187 and quantified. Equal amounts of mRNA were used to generate cDNA using a QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed with NF-kB2/p100 primers (Mm00479807), other FAM-labeled probes, and TaqMan Universal Master mix using a StepOne real-time PCR system (Applied Biosystems). The relative p100 mRNA fold induction was calculated relative to 18S rRNA. Propidium iodide staining assay Splenic AA4.1– mature or AA4.1+ immature B cells from wild-type or Bcl10-deficient mice were cultured at a density of 2 3 106 cells/ml with or without BAFF (100 ng/ml) for the indicated times, followed by propidium iodide staining and FACS analysis. FIGURE 1. Bcl10 deficiency impairs the survival of peripheral B cells in response to BAFF. CD93+ (AA4.1+) and CD932 (AA4.12) B cells from BrdU incorporation assay wild-type (+/+) and Bcl10-deficient (2/2) mice were cultured in the ab- The in vivo BrdU labeling assay was performed as described (47). In brief, sence or presence of BAFF. At the indicated time points, cell survival rates mice were injected i.p. with 1 mg BrdU (Sigma-Aldrich) in 0.2 ml PBS at were determined by propidium iodide staining. Data are representative of 12-h intervals for 4 d. Splenocytes from BrdU-treated mice were stained with five independent experiments. anti-B220. Cells were then fixed and stained with anti-BrdU according to the manufacturer’s instructions of an FITC BrdU flow kit (559619; BD Pharmingen). The degree of BrdU positivity in the gated B cells was increased in the spleens derived from Bcl10-deficient mice relative analyzed by FACS. to those from wild-type animals (Fig. 2A, 2B). In contrast, the Downloaded from TUNEL assay percentage and number of splenic An1 anergic B cells (B220+ CD93+CD23+IgMlo) was drastically reduced in Bcl10-deficient Splenocytes were stained with a combination of fluorescence-conjugated Abs to B220, CD93, CD23, and IgM. Cells were then fixed, permeabilized, and relative to wild-type mice (Fig. 2A–C). Thus, Bcl10 deficiency labeled with fluorescein-conjugated dUTP according to the manufacturer’s results in a drastic reduction in self-reactive anergic B cells. instructions (in situ cell death detection kit, 11-684-795-910; Roche). The degree of TUNEL positivity in the gated B cell subpopulations was analyzed Bcl10 deficiency specifically increases anergic B cell apoptosis

by FACS. in vivo http://www.jimmunol.org/ Next, we examined the rate of apoptosis in mutant An1 anergic Results B cells. We stained splenocytes from wild-type or Bcl10-deficient Bcl10 deficiency severely impairs BAFF-mediated B cell mice with anti-B220, anti-CD93, anti-IgM, and anti-CD23 and then survival analyzed them for apoptosis by TUNEL assay. Bcl10-deficient An1 All peripheral B cells require signals from both the BCR and BAFF- anergic B cells had an increased rate of apoptosis relative to that of R for their survival (20–25). Self-reactive anergic B cells display the corresponding wild-type B cells (Fig. 2D). In contrast, Bcl10- even greater dependence on BAFF-R signaling for their survival deficient T1, T2, and FO B cells had similarly low rates of apo- (25). In present study, we examined the role of Bcl10 in BAFF- ptosis relative to those of the corresponding wild-type B cell mediated B cell survival. Splenic CD93+ B cells, which contain T1, subpopulations, as previously reported (Fig. 2D) (47). These data by guest on September 30, 2021 T2, and An1 anergic subpopulations, and CD932 B cells, which are demonstrate that Bcl10 deficiency specifically impairs the survival mature follicular (FO) and marginal zone B cells, were isolated of An1 anergic B cells in vivo. from wild-type and Bcl10-deficient mice. The cells were cultured in Reduced Bcl10-deficient anergic B cells in IgHELsHEL mouse the absence or presence of BAFF, and cell viability was determined model at various time points. In the absence of BAFF, both wild-type and Bcl10-deficient CD93+ and CD932 B cells underwent apoptosis To further investigate a role of Bcl10 in BAFF-mediated survival of HEL over time, although more mutant cells than corresponding wild-type An1 anergic B cells, Bcl10-deficient mice were crossed with Ig cells died after the initial time point (Fig. 1). As expected, addition transgenic mice, which bear rearranged H and Igk L chain genes of BAFF markedly rescued both wild-type CD93+ and CD932 encoding a BCR that specifically recognizes HEL (16). sHEL HEL B cells from apoptosis (Fig. 1). Importantly, CD93+ Bcellsfrom induces wild-type Ig transgenic B cells to become anergic (16, Bcl10-deficient mice displayed no survival response to BAFF (Fig. 19). In the absence of self-Ag sHEL, the spleens from Bcl10- HEL 1, right). Although BAFF increased viability of Bcl10-deficient deficient Ig transgenic mice displayed a slight reduction of to- CD932 B cells, this was markedly reduced compared with wild- tal splenic B cells compared with wild-type controls (Fig. 3A, 3B). type CD932 B cells (Fig. 1, left). Thus, BAFF-mediated imma- FACS analysis of splenocytes with B220, CD93, IgM, and CD23 ture B cell survival is entirely Bcl10-dependent, whereas mature staining showed that the spleens from both wild-type and Bcl10- HEL B cells only partially depend on Bcl10 for their survival. deficient Ig transgenic mice had a large population of FO mature B cells, a small population of T1 B cells, a moderate population of Bcl10 deficiency causes drastic reduction in anergic B cells T2 B cells, and no An1 anergic B cells (Fig. 3C). As expected, Prior studies have shown that the survival function of BAFF is chronic exposure of wild-type IgHEL B cells to sHEL induced self- particularly important for self-reactive anergic B cells (22–25). Based reactive transgenic B cells into An1 anergic B cells (Fig. 3C, second on our finding that Bcl10-deficient B cells survived poorly in re- row) and reduced the population of splenic B cells in wild-type sponse to BAFF, we examined the effects of loss of Bcl10 on the IgHELsHEL relative to wild-type IgHEL transgenic mice (Fig. 3A, survival of anergic B cell population. In the spleen, B220+CD932 3B). In contrast, in the absence of Bcl10, sHEL drastically reduced cells are largely mature FO B cells (B220+CD932CD23+IgMlo), splenic B cells, especially An1 anergic B cells, in the spleens of whereas B220+CD93+ cells contain T1 (B220+CD93+CD232IgMhi), Bcl10-deficient relative to wild-type IgHELsHEL mice (Fig. 3). T2 (B220+CD93+CD23+IgMhi), and T3-type An1 anergic (B220+ These results confirm that Bcl10 deficiency results in a drastic re- CD93+CD23+IgMlo) populations (19). Previous studies have shown duction of An1 anergic B cells in the well-defined IgHELsHEL that FO B cells were reduced in the spleens derived from Bcl10- transgenic model of B cell anergy. deficient relative to wild-type mice (47). In this study we found that The severe reduction of the An1 anergic B cells in Bcl10-deficient T1 B cells were slightly increased and T2 B cells were markedly IgHELsHEL mice led us to examine whether the reduction is due to 5188 Bcl10 IN BAFF-INDUCED NF-kB ACTIVATION

transgenic mice. Overall, these data demonstrate that in the absence of self-Ag, Bcl10-deficient peripheral B cells can usually survive in vivo, whereas in the presence of self-Ag, self-reactive Bcl10- deficient B cells undergo apoptosis instead of anergy. Bcl10 deficiency impairs BAFF-induced canonical NF-kB activation Owing to a critical role for BAFF in anergic B cell survival, Bcl10- dependent mechanisms that contribute to BAFF-R signaling were investigated. Bcl10 deficiency could reduce the expression of BAFF-R on B cells, resulting in an impairment of the ability of BAFF to support B cell survival. To examine this possibility, we compared BAFF-R expression on wild-type and Bcl10-deficient splenic B cells at different maturation stages. Based on expression of IgD and IgM, splenic B cells can be divided into IgMhi IgD2 (T1), IgMhiIgDhi (T2), and IgMloIgDhi (FO and An1) B cells (53). FACS analysis demonstrated that subpopulations of Bcl10-deficient B cells expressed comparable levels of BAFF-R

relative to corresponding subsets of wild-type B cells (Fig. 5A). Downloaded from Thus, Bcl10 deﬁciency has no apparent effect on the expression of BAFF-R. The other possible explanation for the impaired ability of BAFF to support the survival of Bcl10-deﬁcient B cells is that Bcl10 is required for BAFF-R signaling. NF-kB activation by BAFF-R is es-

sential for B cell survival (22, 30). BAFF-R can induce canonical http://www.jimmunol.org/ NF-kB activation through IKKb-dependent degradation of IkBa (39, 40). To study the potential role of Bcl10 in BAFF-induced canonical NF-kB activation, we first examined whether Bcl10 interacts with IKKb upon BAFF stimulation. Three independent coimmunoprecipitation analyses demonstrated that BAFF stimula- FIGURE 2. Marked reduction of anergic B cells in Bcl10-deficient mice tion induced the association of Bcl10 with IKKb in wild-type and increased apoptosis of the mutant anergic B cells. (A) Splenic B cell splenic B cells, similar to anti–IgM-induced BCR engagement subpopulations. Splenocytes from wild-type and Bcl10-deficient mice were (Fig. 5B). In contrast, BAFF-induced association of Bcl10 with stained with Abs to B220, CD93, IgM, and CD23. In B220+CD93+-gated IKKb was absent in Bcl10-deficient splenic B cells (Fig. 5B, right). by guest on September 30, 2021 cells, T1 (CD232IgMhi), T2 (CD23+IgMhi), and An1 anergic (CD23+IgMlo) Of note, Bcl10 constitutively associated with MALT1 without B cells are shown. Percentages indicate B cells in the gated B220+ pop- stimulation, as previously described (54) (Fig. 5B, left). Thus, ulation. (B) Bar graphs show the percentages of T1, T2, and An1 anergic B Bcl10 signaling complex recruits IKKb upon BAFF stimulation. + C cellsinthegatedB220 population. ( ) Bar graphs show the numbers of Next, we examined whether Bcl10 is involved in BAFF-induced An1 anergic B cells in the spleens of wild-type and Bcl10-deficient mice. canonical NF-kB activation by assessing IKKb-dependent IkBa (D) Apoptosis of splenic B cell subpopulations. Bar graphs show the phosphorylation at Ser32/36.WecomparedIkBa phosphorylation degree of TUNEL labeling in T1, T2, An1 anergic, and FO B cells of wild-type and Bcl10-deficient mice. Data shown are obtained from at in wild-type and Bcl10-deficient splenic mature B cells following least seven (A, B, D)orfour(C) mice in each group. Error bars show 6 BAFF stimulation. BAFF-induced IkBa phosphorylation was SD. *p , 0.01, **p =0.02. markedly reduced in Bcl10-deficient relative to wild-type cells (Fig. 5C). Thus, Bcl10 deficiency impairs BAFF-induced IKKb activation. increased apoptosis of the mutant cells. TUNEL assay demon- Furthermore, we investigated whether Bcl10 is required for strated that the apoptosis rates of splenic B cells were markedly BAFF-induced activation of the canonical NF-kB by gel mobility increased in Bcl10-deficient relative to wild-type IgHELsHEL shift assays. As previously reported (39, 40), canonical NF-kB double-transgenic mice (Fig. 4A, 4B). Of note, the apoptosis rates activation by BAFF stimulation occurred at an early time point of splenic B cells from both wild-type and mutant IgHEL single- (Fig. 5D) and involved the formation of p50/c-Rel heterodimer transgenic mice were equally low (Fig. 4A, 4B). (Fig. 5E) in wild-type mature B cells. However, this canonical NF-kB We also examined whether impaired cell proliferation contrib- activation by BAFF was severely impaired in Bcl10-deficient mature utes to the drastic reduction of anergic B cells in mutant IgHELsHEL B cells (Fig. 5D, 5E). Mutant B cells only displayed basal levels mice. In vivo BrdU labeling assay demonstrated that the BrdU of transcriptionally inactive p50/p50 homodimers, which were not labeling rates of splenic B cells were reduced in Bcl10-deficient elevated by BAFF stimulation (Fig. 5E). Taken together, these relative to wild-type IgHEL single-transgenic mice (Fig. 4C, 4D). data demonstrate that Bcl10 is critical for BAFF-induced canon- However, the in vivo BrdU labeling rates of splenic B cells were ical NF-kB activation in B cells. comparable between Bcl10-deficient and wild-type IgHELsHEL double-transgenic mice (Fig. 4C, 4D). Of note, splenic B cells in Bcl10 deficiency indirectly impairs BAFF-induced k Bcl10-sufficient and Bcl10-deficient IgHELsHEL double-transgenic noncanonical NF- B activation mice were mainly anergic (Fig. 3C). Compared to wild-type double- Previous studies have shown that BCR-mediated canonical NF-kB transgenic mice, mutant double-transgenic mice had a dramatic re- activity contributes to the basal as well as induced expression of duction of these anergic cells (Fig. 3A, 3C). Thus, Bcl10 deficiency NF-kB2/p100, which is a critical substrate in BAFF-mediated mainly impairs the in vivo survival of anergic B cells in IgHELsHEL noncanonical RelB/p52 activation and B cell survival (55, 56). The Journal of Immunology 5189 Downloaded from

FIGURE 3. Dramatic reduction of Bcl10-deﬁcient anergic B cells in IgHELsHEL mouse model. Splenocytes from Bcl10+/+ IgHEL, Bcl102/2 IgHEL, Bcl10+/+ IgHELsHEL, and Bcl102/2 IgHEL sHEL transgenic mice were stained with Abs to B220, IgM, CD93, and CD23. (A) FACS analysis with B220 and IgM staining of lymphocytes. Percentages indicate B220+ cells in the gated lymphoid populations. (B) Bar graphs show the percentages (upper) and numbers (lower) of total splenic B cells in the spleens of the indicated mice. (C) Splenic B cell subpopulations. Upper, FACS analysis with B220 and CD93 + + staining of lymphocytes; middle, FACS analysis with IgM and CD23 staining of B220 CD93 -gated cells; lower, FACS analysis with IgM and CD23 http://www.jimmunol.org/ staining of B220+CD932-gated cells. Percentages indicate cells in the gated lymphoid populations. (D) Bar graphs show the numbers of CD93+ An1 anergic B cells in the spleens of the indicated mice. Data shown are obtained from 11 (A, B), 3 (C), or 4 (D) mice in each group. Error bars show 6 SD. *p , 0.01, **p = 0.01.

Bcl10 deficiency disrupts BCR-mediated NF-kB activation (46, It is well established that constant BAFF exposure induces non- 47). Our Western blot analysis of the expression of the NF-kB canonical NF-kB activation through a relatively well-defined path- family members revealed that the basal level of NF-kB2/p100, but way that requires processing of NF-kB2/p100 into p52. The p52 not other NF-kB members, was reduced in Bcl10-deficient relative then dimerizes with RelB to form p52/RelB heterodimer (35, 36). to wild-type splenic mature B cells (Fig. 6A). However, the NF- Our results showed that induction of NF-kB DNA binding activity by guest on September 30, 2021 kB2/p100 protein was efficiently processed into p52 in the by constant BAFF exposure was severely reduced in Bcl10-deficient presence of BAFF in both wild-type and Bcl10-deficient mature relative to wild-type B cells (Fig. 6D). B cells, as indicated by reduction in NF-kB2/p100 protein (Fig. These NF-kB DNA–binding complexes in wild-type B cells were 6B). Consistent with reduced NF-kB2/p100 protein in the Bcl10- supershifted strongly by anti-p50 and slightly by anti–c-Rel or anti- deficient B cells, BAFF-induced NF-kB2/p100 mRNA expression RelB, whereas the supershift with anti-RelA/p65 or anti-p52 was also reduced in Bcl10-deficient relative to wild-type immature was least prominent (Fig. 6E). However, anti-RelA/p65 and and mature B cells (Fig. 6C). Thus, lack of Bcl10 reduces basal anti-p52 reduced intensity of BAFF-induced NF-kB bands (Fig. and BAFF-induced expression of NF-kB2/p100 in B cells. This 6E). These results suggest that in addition to the expected p52/ limits the p100 substrate necessary for the optimal activation of RelB heterodimer (35, 36), p50/RelA and p50/c-Rel heterodimers noncanonical NF-kB in Bcl10-deficient B cells. were also formed in response to BAFF.

FIGURE 4. Increased apoptosis and normal proliferation of anergic B cells in Bcl10-deﬁcient IgHELsHEL mice. (A) TUNEL labeling of splenic B cells. Splenocytes from Bcl10+/+ IgHEL, Bcl102/2 IgHEL, Bcl10+/+ IgHELsHEL, and Bcl102/2 IgHELsHEL transgenic mice were stained with anti-B220 Abs. Then, the degree of TUNEL labeling in B220+ cells was determined by FACS analysis. Percentages indicate TUNEL+ cells in the gated B220+ cells. (B) Statistical analysis of the percentages of TUNEL+ cells from (A). (C) BrdU incorporation in splenic B cells. BrdU was injected into Bcl10+/+ IgHEL, Bcl102/2 IgHEL,Bcl10+/+ IgHELsHEL, and Bcl102/2 IgHELsHEL transgenic mice. Splenocytes from the mice were stained with anti-B220 Abs, followed by BrdU staining. Percentages indicate BrdU+ cells in the gated B220+ cells. (D) Statistical analysis of the percentages of BrdU+ cells from (C). Data shown are obtained from at least ﬁve (A, B)orseven(C, D) mice in each group. Error bars show 6 SD. *p , 0.01. 5190 Bcl10 IN BAFF-INDUCED NF-kB ACTIVATION

FIGURE 5. Bcl10 deficiency impairs BAFF-induced canonical NF-kB activation. (A) Expression of BAFF-R. Splenocytes from wild-type and Bcl10- deficient mice were stained with Abs to CD19, IgM, IgD, and BAFF-R. Expression levels of BAFF-R on total splenic (CD19+), T1 (IgMhiIgD2), T2 (IgMhi Downloaded from IgDhi), and FO (IgMloIgDhi) B cells were measured by FACS analysis. (B–E) Splenic mature B cells (CD932) were isolated from wild-type and Bcl10- deficient mice. (B) BAFF-induced association of Bcl10 with IKKb. Wild-type or Bcl10-deficient mature B cells were stimulated with BAFF or anti-IgM for the indicated times. Cell lysates were immunoprecipitated with anti-Bcl10–conjugated Sepharose beads, followed by Western blotting analysis with the indicated Abs. (C) BAFF-induced IkBa phosphorylation. Cells were pretreated with MG132 and subsequently stimulated with BAFF for the indicated times. Cell lysates were subjected to direct Western blotting analysis with anti–phospho-IkBa or anti-actin Abs. (D and E) BAFF-induced canonical activation of NF-kB. Cells were stimulated with (+) or without (2) BAFF for 3 h and total cell lysates were subjected to NF-kB gel mobility shift (D) and E D E A C E Ab supershift ( ) analysis. Arrows indicate the position of NF-kB complexes ( ) and p50/p50 homodimers ( ). Data are representative of three ( , – )or http://www.jimmunol.org/ two (B) independent experiments.

In contrast, the NF-kB DNA–binding complexes in Bcl10- agreement with reduced basal p100 protein levels combined with deficient B cells were supershifted or reduced in intensities by an inability to transcriptionally upregulate p100. Nonetheless, the all Abs used except anti-p52 (Fig. 6E). These results are consistent limiting amounts of NF-kB2/p100 available in Bcl10-deficient with reduced canonical NF-kB activation (p50/RelA and p50/c- B cells were efficiently processed into p52 and subsequently Rel heterodimers) in Bcl10-deficient B cells. Furthermore, a se- translocated into nucleus (Fig. 6B). Taken together, these data vere reduction in the formation of p52/RelB heterodimer was in demonstrate that Bcl10 deficiency severely impairs the formation by guest on September 30, 2021

FIGURE 6. Bcl10-deficient splenic B cells express reduced levels of p100, indirectly affecting BAFF-induced noncanonical NF-kB activation. (A) Expression of NF-kB/Rel family members. Protein levels of NF-kB/Rel family members in total cellular extracts from splenic mature B cells were determined by direct Western blotting analysis with the indicated Abs. Densitometric analysis of protein bands was performed and normalized to actin loading control. Levels of each NF-kB protein in Bcl10-deficient and wild-type B cells were compared by giving wild-type an arbitrary value of 1. The bar graph shows the relative p100 protein levels calculated from three independent experiments. p , 0.01. (B) BAFF-induced NF-kB2/p100 processing and nuclear translocation. Following 16 h culture of splenic mature B cells with or without BAFF, cytoplasmic and nuclear extracts were prepared and subjected to Western blotting with the indicated Abs. GAPDH was used as cytoplasmic protein loading control and YY1 as nuclear protein loading control. (C) BAFF- induced NF-kB2/p100 mRNA expression. Splenic immature (CD93+) and mature (CD932CD23+) B cells were either stimulated with BAFF or left nonstimulated for 16 h. NF-kB2/p100 mRNA expression was determined by quantitative real-time PCR. Relative fold induction in response to BAFF was normalized to 18S rRNA and calibrated to nonstimulated sample for the corresponding genotype. (D and E) BAFF-induced noncanonical activation of NF-kB. Splenic mature B cells were constantly stimulated with BAFF for 16 h and total cell lysates were subjected to NF-kB gel mobility shift (D) and Ab supershift (E) analysis. Arrows indicate the position of NF-kB complexes. (F) BAFF-induced ERK and Akt activation. Splenic mature B cells were stimulated with BAFF for the indicated times and cell lysates were subjected to direct Western blotting analysis with the indicated Abs. Data are representative of three (A, B, D, E) or two (F) independent experiments, or data are representative of at least three experiments that used one animal from each genotype per experiment (C). The Journal of Immunology 5191 of not only p50/RelA and p50/c-Rel complexes, typically activated by the canonical NF-kB pathway, but also p52/RelB complexes, typically activated by the noncanonical NF-kB pathway. The impairment of noncanonical NF-kB activation in Bcl10- deficient B cells is likely due to the reduction of NF-kB2/p100, which is in part regulated by the canonical NF-kB pathway. In addition to the NF-kB pathway, BAFF stimulation promotes B cell survival by downregulating the proapoptotic molecule Bim via ERK activation as well as by activating the prosurvival kinase Akt (57, 58). To address the role that Bcl10 plays in these pathways, we examined whether Bcl10 is involved in BAFF-induced activation of ERK and Akt. Splenic mature B cells were isolated from wild-type and Bcl10-deficient mice and then stimulated with BAFF. The activation of ERK was evaluated by immunoblotting with Abs that detect phosphorylation of Thr202//Tyr204 within ERK1 and Thr185//Tyr187 within ERK2. BAFF-induced activation of ERK FIGURE 7. Bcl10 deficiency impairs BAFF-induced expression of NF- was comparable in Bcl10-deficient and wild-type B cells (Fig. 6F). kB target gene Bcl-xL. Splenic mature B cells were isolated from wild- Additionally, the activation of Akt was evaluated by immunoblot- type and Bcl10-deficient mice and stimulated with BAFF for the indicated times. Cell lysates were subjected to direct Western blotting analysis with ting with Abs that detect phosphorylation of Ser473 within Akt. Downloaded from the indicated Abs. Data from two independent experiments are shown. BAFF-induced activation of Akt was biphasic. The initial activation, which occurred 5–10 min poststimulation, was reduced to basal level 30 min later, followed by a second phase of activation at of anergy. The IgHEL single transgene did not appear to affect T1 24 h (Fig. 6F). This biphasic activation of Akt by BAFF was not and T2 B cells and only slightly reduced mature FO B cells in affected in Bcl10-deficient mature B cells (Fig. 6F). Thus, Bcl10 is Bcl10-deficient relative to wild-type control mice, indicating that

not required for BAFF-induced activation of ERK and Akt. Bcl10 function was not critical for the survival of peripheral http://www.jimmunol.org/ B cells in the absence of self-Ag. In IgHELsHEL double-transgenic Bcl10 deficiency impairs BAFF-dependent NF-kB target gene model, soluble self-Ag, sHEL, drove self-reactive B cells into expression anergy in both wild-type and mutant mice. Anergic B cells that The activation of the NF-kB pathway by BAFF stimulation ulti- depend more heavily on BAFF underwent more apoptosis in the mately upregulates the expression of antiapoptotic genes, such as absence of Bcl10. Thus, the BAFF-R/Bcl10/NF-kB signaling axis Bcl-xL (22, 30). To confirm that Bcl10 plays an important role in contributes to the establishment of peripheral B cell tolerance by BAFF-mediated survival of B cells through the NF-kB pathway, maintaining anergic B cells and preventing these B cells from self- we examined BAFF-induced upregulation of the prosurvival Ag–induced elimination. NF-kB target gene, Bcl-xL, in Bcl10-deficient B cells. Following BAFF can induce canonical and noncanonical NF-kB activation by guest on September 30, 2021 BAFF stimulation, the level of Bcl-xL proteins was markedly pathways, both of which are required for B cell survival (35, 59). upregulated in wild-type B cells (Fig. 7). In contrast, the upreg- Whereas BAFF-R–induced activation of the noncanonical NF-kB ulation of Bcl-xL proteins was abolished in Bcl10-deficient B cells pathway is relatively well studied, the membrane-proximal sig- (Fig. 7). Of note, the amount of Bcl-2 proteins remained unaltered naling cascades upstream of the canonical pathway remain unclear. before and after BAFF stimulation in both wild-type and Bcl10- Our current studies demonstrate that Bcl10 directly participated in deficient B cells (Fig. 7). Therefore, Bcl10 is required for BAFF- BAFF-induced activation of canonical NF-kB via mechanisms induced expression of Bcl-xL, a prosurvival gene of the Bcl-2 involving direct association of Bcl10 with IKKb. This results in family that is regulated by NF-kB. the activation of the IKK complex consisting of IKKa, IKKb, and NEMO. Our observation that noncanonical NF-kB activation by Discussion BAFF was impaired in Bcl10-deficient mice suggests a role for Previous studies have demonstrated that the survival of all peripheral Bcl10 in the regulation of this NF-kB pathway as well. A direct B cells as well as maintenance of anergic B cells depends on signals role for Bcl10 in activation of the noncanonical pathway was from both the BCR and BAFF-R (20–24). Our findings demonstrate ruled out by our data showing that Bcl10 deficiency did not af- that Bcl10 was essential for anergic B cell survival. This Bcl10 fect NF-kB2/p100 to p52 processing. Instead, this defect was function was apparent in the diverse repertoire as well as IgHEL foundtobeanindirecteffectofBcl10deficiencyontheex- system. Our findings further suggest that this was likely the con- pression of NF-kB2/p100. Thus, our results suggest that reduced sequence of a failure to activate both canonical and noncanonical availability of NF-kB2/p100 limited the extent of noncanonical NF-kB pathways; Bcl10 directly contributed to BAFF-mediated NF-kB activation in Bcl10-deficient B cells. activation of the canonical NF-kB pathway and indirectly dis- It is well known that Bcl10 forms a ternary complex with rupted the BAFF-mediated noncanonical NF-kB pathway by re- CARMA1 and MALT1 (CBM) to couple PKC activity to IKK ducing the availability of p100. Thus, Bcl10 plays a critical role in complex activation during BCR-induced canonical NF-kB acti- the peripheral B cell anergy in part by mediating BAFF-R signaling. vation (48–51). Our data demonstrating a requirement for Bcl10 in Our data demonstrate a dramatic reduction of anergic B cells in the activation of the canonical pathway suggest that the CBM Bcl10-deficient mice relative to wild-type controls. Consistently, complex is required for BAFF-induced activation of the IKK Bcl10-deficient An1 anergic B cells displayed increased rate of complex. This hypothesis is supported by our finding that Bcl10 is apoptosis in vivo relative to the corresponding wild-type B cells, constitutively associated with MALT1, suggesting that Bcl10/ whereas rates of apoptosis were similar when T1, T2, or FO B cell MALT1 partner with CARMA1 to form the CBM complex to ac- populations were compared. The finding that Bcl10 deficiency tivate the IKK complex during BAFF-R signaling. This possibility primarily affected the survival of anergic B cells with diverse remains to be investigated, and the role of MALT1 in the activa- repertoire was recapitulated in the Ig transgenic IgHELsHEL model tion of the canonical NF-kB pathway remains unknown. However, 5192 Bcl10 IN BAFF-INDUCED NF-kB ACTIVATION a recent study has demonstrated that MALT1 regulates BAFF- 3. Rajewsky, K. 1996. Clonal selection and learning in the antibody system. Nature 381: 751–758. induced noncanonical NF-kB activation in B cells. MALT1 defi- 4. Nemazee, D. 2000. Receptor selection in B and T lymphocytes. Annu. Rev. ciency impairs BAFF-induced phosphorylation and processing of Immunol. 18: 19–51. NF-kB2/p100, as well as RelB nuclear translocation, suggesting 5. Healy, J. I., and C. C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 16: 645–670. a direct role for MALT1 in the noncanonical NF-kB activation 6. Radic, M. Z., and M. Zouali. 1996. Receptor editing, immune diversification, (60). Additionally, the role of CARMA1 in BAFF-induced ca- and self-tolerance. Immunity 5: 505–511. nonical NF-kB activation is also unknown. Our findings suggest 7. Sandel, P. C., and J. G. Monroe. 1999. Negative selection of immature B cells by receptor editing or deletion is determined by site of antigen encounter. Immunity that the Bcl10-containing CBM complex mediates the canonical 10: 289–299. NF-kB pathway. Our results lay the foundation for further studies 8. Nossal, G. J. 1994. Negative selection of lymphocytes. Cell 76: 229–239. to determine the contribution of CARMA1 and MALT1 to BAFF- 9. Radic, M. Z., J. Erikson, S. Litwin, and M. Weigert. 1993. B lymphocytes may escape tolerance by revising their antigen receptors. J. Exp. Med. 177: 1165– induced canonical NF-kB activation. 1173. The membrane-proximal steps of the BCR signaling cascade that 10. Gay, D., T. Saunders, S. Camper, and M. Weigert. 1993. 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