Nuclear Export of the NF-κB Inhibitor IκBα Is Required forProper B Cell and Secondary Lymphoid Tissue Formation

Shelly M. Wuerzberger-Davis1,8, Yuhong Chen2,8, David T. Yang3, Jeffrey D. Kearns4, PaulW. Bates1,9, Candace Lynch4, Nicholas C. Ladell1, Mei Yu2,5, Andrew Podd2,6, Hu Zeng2,5,Tony T. Huang7, Renren Wen2, Alexander Hoffmann4, Demin Wang2,5,6,*, and ShigekiMiyamoto1,*

1McArdle Laboratory for Cancer Research, Department of Oncology, University of WisconsinCarbone Cancer Center, University of Wisconsin-Madison, 6159 Wisconsin Institute for MedicalResearch, 1111 Highland Avenue, Madison, WI 53705, USA2Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53226, USA3Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, B4/259Clinical Sciences Center, 600 Highland Avenue, Madison, WI 53792, USA4Signaling Systems Laboratory, Department of Chemistry and Biochemistry, University ofCalifornia, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA5State Key Laboratory of Pharmaceutical Biotechnology, College of Life Science, NanjingUniversity, Nanjing 210093, P. R. China6Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee,WI 53226, USA7Department of Biochemistry, New York University, New York, NY 10016, USA

SUMMARYThe N-terminal nuclear export sequence (NES) of inhibitor of nuclear factor kappa B (NF-κB)alpha (IκBα) promotes NF-κB export from the cell nucleus to the cytoplasm, but the physiologicalrole of this export regulation remains unknown. Here we report the derivation and analysis ofgenetically targeted mice harboring a germline mutation in IκBα NES. Mature B cells in themutant mice displayed nuclear accumulation of inactive IκBα complexes containing a NF-κBfamily member, cRel, causing their spatial separation from the cytoplasmic IκB kinase. Thisresulted in severe reductions in constitutive and canonical NF-κB activities, synthesis of p100 andRelB NF-κB members, noncanonical NF-κB activity, NF-κB target gene induction, andproliferation and survival responses in B cells. Consequently, mice displayed defective B cellmaturation, antibody production, and formation of secondary lymphoid organs and tissues. Thus,IκBα nuclear export is essential to maintain constitutive, canonical, and noncanonical NF-κBactivation potentials in mature B cells in vivo.

©2011 Elsevier Inc.*Correspondence: demin.wang@bcw.edu (D.W.), smiyamot@wisc.edu (S.M.).8These authors contributed equally to this work9Present address: Department of Biology, University of Minnesota, Duluth, MN 55812, USASUPPLEMENTAL INFORMATIONSupplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article onlineat doi:10.1016/j.immuni.2011.01.014.

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Published in final edited form as:Immunity. 2011 February 25; 34(2): 188–200. doi:10.1016/j.immuni.2011.01.014.

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INTRODUCTIONThe NF-κB-Rel family of transcription factors regulates multiple physiologic processes,including innate and adaptive immunity and various stress responses (Ghosh and Hayden,2008; Perkins, 2007). In mammals, this consists of five members, RelA (p65), cRel, RelB,NFkB1 (p50), and NFkB2 (p52), which form dimers, such as the most widely expressedRelA:p50 or more tissue-restricted cRel homo- and heterodimers. A key feature of NF-κBdimers is their cytoplasmic localization as inactive complexes while bound to members ofthe inhibitor of NF-κB (IκB) family, such as IκBα and IκBβ. Activation of NF-κB requiresits release from IκB to allow nuclear migration and target gene regulation. “Canonical”activation involves the activation of the cytoplasmic IκB kinase (IKK) complex composedof IKKα (IKK1), IKKβ (IKK2), and IKKγ (NF-κB essential modulator, NEMO) thatinduces phosphorylation-regulated degradation of IκB, releasing NF-κB dimers to thenucleus. This activation pathway is induced by a variety of extracellular stimuli or stressconditions and is principle in many NF-κB activation processes (Ghosh and Hayden, 2008;Perkins, 2007). An alternative “noncanonical” pathway exists, where the precursor of p52,p100, is phosphorylated by the IKKα complex, without the need for IKKβ and NEMO. Afterphosphorylation, p100 is processed to selectively activate a RelB:p52 heterodimer inresponse to specific inducers. RelB:p52 complexes do not associate with canonical IκBproteins and therefore are not directly regulated by them. The noncanonical pathway iscritical for lymphoid organ development and immune cell development, among others(Hoffmann and Baltimore, 2006; Sen, 2006).

Classically, IκB is thought to mask the nuclear localization sequence (NLS) of RelA toprevent its nuclear entry, thereby “sequestering” NF-κB in the cytoplasm (Baeuerle andBaltimore, 1988). This mode of regulation appears to be the case for complexes containingIκBβ (Huang et al., 2000; Malek et al., 2001; Tam et al., 2001). However, studies employingthe nuclear export inhibitor leptomycin B (LMB) provide contrasting evidence thatRelA:IκBα, cRel:IκBα, and RelA:IκBε complexes shuttle between the cytoplasm and thenucleus in their inactive state (Carlotti et al., 2000; Huang et al., 2000; Johnson et al., 1999;Malek et al., 2001; Tam et al., 2000). In support of this dynamic “nucleocytoplasmicshuttling” model, RelA:p50:IκBα cocrystal structures indicate that IκBα masks the NLS ofRelA but spares that of p50 (Huxford et al., 1998). Moreover, p50 NLS is found to becritical for nuclear import of RelA:p50:IκBα complexes (Huang et al., 2000; Malek et al.,2001; Tam et al., 2001). An alternative model has also been implicated in which NF-κB andIκBα complexes enter the nucleus separately but exit together (Carlotti et al., 2000; Tam etal., 2000). The mechanism of nuclear export of the complexes also appears intricate,possibly involving multiple distinct nuclear export sequences (NESs) present on IκBα, IκBε,and RelA (Huang et al., 2000; Johnson et al., 1999; Malek et al., 2001; Tam et al., 2000).Interestingly, other NF-κB family members, such as cRel and p50, do not contain NESmotifs in their sequences, suggesting that their export depends on a nuclear export functionprovided primarily by IκBα. However, these studies employed cell culture models oftenutilizing LMB and/or transient overexpression of respective proteins, so the physiologicalimportance of this NES-mediated shuttling mechanism has been questioned (Ghosh andKarin, 2002). Indeed, there has not been any direct in vivo study to evaluate thephysiological role of nuclear export of any of the NF-κB:IκB complexes and mechanismsimplicated.

To address this question, we created a genetically targeted mouse model harboring agermline mutation in the N-terminal NES of IκBα (Huang et al., 2000). Here, we havedescribed the mechanistic and phenotypic characterization of the mutant mice and cellsderived from them. Our results reveal a surprising finding that the nuclear export functionmediated by IκBα N-NES is essential for basal, canonical, and noncanonical NF-κB

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activation in B lymphocytes, maturation of B cells, and formation of several secondarylymphoid tissues. Our study reveals insight into important physiological and cell type-selective functions of nuclear export regulation of the NF-κB-IκB signaling system in vivo.

RESULTSGeneration of NfkbiaNES/NES Genetically Targeted Mice

We created NfkbiaNES/NES mice harboring a triple point mutation in the N-terminal NES ofIκBα, M45A, L49A, and I52A (Huang et al., 2000) in the germline (Figures S1A and S1Bavailable online). The heterozygous Nfkbia+/NES mice were backcrossed with C57BL/6Jmice for 5–7 generations. Homozygous mutant mice were also bred to each other. Thesestudies demonstrated that both male and female NfkbiaNES/NES mice were born in aMendelian ratio, fertile, and indistinguishable from their wild-type (WT) counterparts basedon size, weight, or general appearance at sexual maturity (not shown). Allele-specific RT-PCR analysis of total RNA (Figure S1C) and immunoblot analysis of total protein extractsfrom several tissues (Figure S1D) demonstrated the expression of the mutant gene and theprotein in NfkbiaNES/NES mice. The migration of the mutant protein in sodium dodecylsulfate polyacrylamide gel electrophoresis was partially retarded compared to WT protein(Figure S1D), a phenomenon also observed when the protein was transiently expressed inHEK293 cells (not shown). This suggests that the property is an intrinsic migration anomalyresulting from the substitution mutations introduced.

Abnormal Formation of Secondary Lymphoid Organs and Tissues in NfkbiaNES/NES MiceA closer inspection revealed that the inguinal lymph nodes were often bilaterally absent inthe mutant mice (Figure 1A) and, when present, they were considerably smaller and showeddisrupted B cell organization (Figures 1B and 1C). Therefore, we next examined otherlymphoid tissues and organs more closely. Although other lymph nodes analyzed (cervical,mesenteric, and lumber) were comparable to WT numbers, intestine-associated Peyer’spatches, mucosal lymphoid organs involved in protection from intestinal microbes as well asproduction of IgA, were markedly reduced in number and size in the mutant mice (Figures1D and 1E). Finally, although the size and weight of spleens were indistinguishable betweenWT and mutant littermates, the organization of B cells and marginal zone (MZ) architecturewere also disrupted in the spleen of the mutant mice (Figures 1F and 1G). In contrast, basedon morphology, weight, and histology, the thymus was indistinguishable between WT andmutant mice (Figure 1H and data not shown). Thus, IκBα N-NES is essential for properformation of several secondary lymphoid organs and tissues.

Impaired Maturation of B Cells in NfkbiaNES/NES MiceWe next assessed the development and maturation of lymphocytes in the bone marrow(BM), spleen, and thymus of NfkbiaNES/NES mice based on cell surface marker expression(Hardy and Hayakawa, 2001). An expanded population of pre-B cells (B220+CD43−IgM−)was observed in mutant mice leading to slightly increased BM B cell numbers, but overalltotal BM cell numbers were comparable between WT and mutant mice (Figures S2A–S2D).Despite the increase in the absolute number of immature B cells (B220+IgM+) inNfkbiaNES/NES mice, the population of mature B cells (B220hiIgM+) in BM was decreased(Figures S2B and S2D). In the spleen, the percentage of T cells was increased and that of Bcells was decreased (Figure 2A), but the number of total Thy1.2+ (Figure 2B) or CD4+ andCD8+ T cells in the spleen was similar in NfkbiaNES/NES mice (Table 1). Similarly, thymic Tcell development was indistinguishable between mutant and WT mice based on CD4 andCD8 staining (Figures 2C and 2D). The difference in the percentage of T and B cells in thespleen was due to the reduction of the number of B220+ B cells (Figure 2B), involvingreductions of the transitional 2 (T2) and follicular (FO), but not transitional 1 (T1) B cell

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fractions (Figures 2E, 2F, and 2I). The MZ B cells (Oliver et al., 1997) were also decreased(Figures 2F–2I). Additionally, the self-renewing mature B1a and B1b B cells that reside inthe peritoneal and pleural cavities (Fagarasan et al., 2000) were largely normal but thepopulation of peritoneal B2 B cells was substantially reduced in NfkbiaNES/NES mice(Figures S2E and S2F). Finally, there was a statistically significant reduction of serum IgA,IgG1, and IgG2b amounts in the mutant mice (Figure 2J). Taken together, we conclude thatthe NES mutation in IκBα impairs B cell maturation, resulting in reduction of T2, FO, andMZ B cells. Moreover, the failure of BM cells derived from NfkbiaNES/NES mice to properlyreconstitute T2, FO, and MZ B cells in irradiated B cell-null Jak3−/− (Figures 3A–3D;Nosaka et al., 1995; Thomis et al., 1995) or Rag2−/− (not shown) mice demonstrated thatthese B cell defects in NfkbiaNES/NES mice were hematopoietically cell intrinsic. The abovephenotypes observed in NfkbiaNES/NES mice were also detected at variable amounts inheterozygous Nfkbia+/NES mice (Table 1), demonstrating that a single copy of the mutantallele had a variably dominant impact on these biological processes.

Diminished Constitutive and Canonical Activation of NF-κB in NfkbiaNES/NES B CellsTo determine whether NF-κB activation was affected in B cells of the mutant mice, weisolated splenic B cells and performed electrophoretic mobility shift assays (EMSA) andsupershift analyses. We found that a constitutive NF-κB complex primarily composed ofcRel and p50 or p52 (Figure 4A, upper complex) was severely reduced in NfkbiaNES/NES Bcells compared to WT cells. When these B cells were exposed to anti-IgM to stimulate Bcell receptor signaling, we also observed activation defects (Figure 4B). Similarly, mutant Bcells were incapable of efficiently activating NF-κB in response to lipopolysaccharide (LPS)(Figure 4C), demonstrating that mutant B cells were defective in canonical NF-κBactivation. Anti-IgM- and LPS-induced degradation of IκBα was less efficient in mutant Bcells (Figures 4D and 4E), despite similar IKK activation as observed after LPS stimulation(Figure 4F) in WT and NfkbiaNES/NES B cells. IκBβ degradation appeared to be induced atcomparable amounts (Figures 4D and 4E). When we examined the subcellular localizationof IκBα by immunofluorescence analysis, it was predominantly localized in the nucleus inmutant B cells (Figures 4G–4I). Similarly, more cRel was found in the nucleus (Figures 4G–4I) even though it did not bind DNA (Figure 4A). Coimmunoprecipitation analysis showedthat IκBα was associated with cRel (Figure 4J). We were unable to obtain similarly highnuclear IκBα and cRel amounts in mutant B cells by a cell fractionation approach, probablybecause of technical limitations (Figure S3A). In total, the results suggest that spatialseparation between active cytoplasmic IKK and nuclear inactive cRel: IκBα complexesprevented efficient canonical NF-κB activation in NfkbiaNES/NES B cells. Interestingly, RelAsubcellular localization was perturbed to a much smaller extent in B cells (Figures S3B andS3C), possibly because of the presence of a compensatory NES in RelA (Harhaj and Sun,1999; Tam et al., 2001).

Defective Noncanonical NF-κB Activation in NfkbiaNES/NES B CellsIn the above coimmunoprecipitation analysis, we noted that the steady-state amount of cRelwas reduced in mutant B cells compared to WT cells (Figure 4J). The expression of thegenes encoding several NF-κB family members is controlled by NF-κB in B cells because ofthe presence of κB elements in their promoters (Grumont and Gerondakis, 1994; Perkins,2007). Accordingly, expression of cRel, RelB, p105 (p50), and p100 (p52) in the mutant Bcells was reduced compared to the WT littermate control (Figure 5A). Expression of RelA inmutant B cells was also slightly reduced. Quantitative real-time polymerase chain reactionanalysis showed that amounts of transcripts encoding cRel, RelB, and p100 (p52) wereconsiderably lower in the NfkbiaNES/NES B cells (Figure 5B). Moreover, although thetranscript of the mutant Nfkbia gene was not statistically significantly different (not shown),the amount of mutant IκBα protein was higher in mutant B cells (Figures 4D, 4E, 4J, and

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5A). This was also evident in Nfkbia+/NES B cells (Figure 5A). This is due to higher stabilityof mutant IκBα protein (Figure 5C), consistent with its resistance to signal-induceddegradation (Figures 4D and 4E). The relative stability of the mutant IκBα would lead to agreater steady-state pool of the mutant as compared to WT IκBα. This could explain whyNfkbia+/NES mice also had considerable defects in B cell maturation and development oflymph nodes and Peyer’s patches (Table 1), functioning as a dominant-suppressive IκBαmutant protein in vivo. Finally, IκBβ was slightly reduced in the mutant B cells (Figures 4D,4E, 4J, and 5A), possibly reflecting the reduced expression of its partner NF-κB proteins inthese cells. Although B cell activating factor-receptor (BAFF-R) expression was normal(Figure S4A) and BAFF-induced p100 processing was evident (Figure 5D), there was asevere defect in activation of BAFF-induced noncanonical NF-κB activation containingRelB (Figure S4B) in NfkbiaNES/NES B cells (Figure 5E). In contrast, basal expression ofp100 and all other NF-κB family members was similar between mouse embryonicfibroblasts (MEFs) derived from NfkbiaNES/NES and WT littermate controls (Figure 5F;Figure S4C). Noncanonical NF-κB activity induced by anti-lym-photoxin beta (LTβ) wasalso similar in these cells (Figure 5G). Canonical activation induced by tumor necrosisfactor alpha (TNF-α) or LPS was also mostly normal in MEFs derived from NfkbiaNES/NES

mice (Figure S4D). Thus, the reduced basal NF-κB family member expression and canonicaland noncanonical NF-κB activation defects seen in mutant B cells were cell type-selectivephenotypes. This was associated with a marked reduction in constitutive activation of cRelcomplexes along with constitutive nuclear cRel:IκBα complexes that were resistant tosignal-induced activation in mature B cells. Taken together, these results demonstrate that anuclear export defect in IκBα caused large-scale perturbations in the capacity of B cells tomaintain NF-κB activation potentials through constitutive, canonical, and noncanonicalpathways.

Defective Proliferation and Survival in NfkbiaNES/NES B CellsDeficiencies in canonical and noncanonical NF-κB activation are associated with cellularproliferation and apoptosis defects in B cells (Ghosh and Hayden, 2008; Vallabhapurapu andKarin, 2009). Consistent with severe canonical and noncanonical NF-κB activation defects,we found that B cells (both AA4.1+ immature and AA4.1− mature) from NfkbiaNES/NES

mice displayed reduced proliferation rates compared to WT cells (Figures 6A and 6B). Thiscorrelated with reduced entry of NfkbiaNES/NES B cells into S and G2-M phases upon anti-IgM+interleukin-4 (IL-4) or LPS stimulation (Figures 6C and 6D). Upon stimulation, moremature B cells from NfkbiaNES/NES mice underwent apoptosis than those from WT cells(Figure 6D). Accordingly, expression of Myc and Bcl2l1 (encoding protein BCLxL), bothNF-κB target genes involved in proliferation and survival, respectively, was markedlyreduced in mutant B cells (Figures 6E and 6F). Thus, IκBα N-NES is also critical for B cellproliferation and survival. In contrast, proliferation of thymic and splenic T cells from bothWT and NfkbiaNES/NES mice in response to anti-CD3, anti-CD3+anti-CD28, or anti-CD3+IL-2 was indistinguishable (Figure 6G). Subcellular localization of mutant IκBα inNfkbiaNES/NES T cells was mostly normal in association with cytoplasmic RelA (FiguresS5A and S5B). A flurry of recent reports demonstrates that cRel plays an essential role inthe development of regulatory T cells by promoting Foxp3 transcription (Deenick et al.,2010; Hori, 2010; Isomura et al., 2009; Long et al., 2009; Ruan et al., 2009; Vang et al.,2010; Visekruna et al., 2010; Zheng et al., 2010), so we also investigated whether regulatoryT cells were affected in the mutant mice. Analysis of a limited number of WT andNfkbiaNES/NES mice showed statistically significant developmental defects of this cell type inthe mutant mice (Figures S5C and S5D). Similarly, cRel and p50 have also been implicatedin the development of CD4+ memory-like T cells (Zeng et al., 2008; Zheng et al., 2003).Likewise, we also found statistically significant reductions of CD4+, but not CD8+,memory-like T cells in the mutant mice (Figures S5E and S5F). Thus, in these T cell

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subsets, cRel activities are probably disrupted in the NfkbiaNES/NES mice. Overall, our datademonstrate that a defect in the IκBα N-NES function causes multiple unexpectedbiochemical and functional perturbations in mature B cells and subsets of T cells andmalformation of multiple secondary lymphoid tissues and organs.

DISCUSSIONIn the present study, we demonstrated that IκBα N-NES-mediated export of NF-κB hasimportant physiological roles, particularly for maturation of B cells and proper formation ofseveral secondary lymphoid organs and tissues. The primary molecular defect appears to bethe abnormal nuclear localization of inactive cRel:IκBα complexes, which makes themresistant to signal-induced IκBα degradation and cRel complex activation in NfkbiaNES/NES

B cells. Because the mutant protein accumulates over the WT protein (because of itsresistance to degradation), it also functions as a dominant-suppressive mutant causingheterozygous Nfkbia+/NES mice to similarly show significant in vivo defects. Consistent withthese findings, some of the phenotypes observed in NfkbiaNES/NES mice were reminiscent ofthose previously described in Rel−/− mice, such as reduced proliferation of splenic B cellsand a reduction of serum IgG1 (Köntgen et al., 1995). Furthermore, NfkbiaNES/NES mice alsodisplayed reductions in FO and MZ B cell populations and serum IgA previously observedin Rel−/−Nfkb1−/− double-mutant mice (Pohl et al., 2002). In contrast, NfkbiaNES/NES miceshowed normal peritoneal B1 B cell development, unlike Rel−/−Nfkb1−/− mice (Pohl et al.,2002). It is likely that the amount of inhibition of cRel:p50 activity or other cRel-associatedcomplexes is not sufficiently inhibited in B1 B cells of NfkbiaNES/NES mice to yieldprofound defects observed in Rel−/−Nfkb1−/− mice.

We also observed severe defects in noncanonical NF-κB activation in NfkbiaNES/NES B cells.This was a surprising finding because IκBα does not bind RelB:p52 heterodimers andtherefore it cannot directly inhibit noncanonical NF-κB activity. Accordingly, BAFF-induced p100 processing to p52 could be readily detected, indicating that the noncanonicalsignaling pathway per se is not defective in NfkbiaNES/NES B cells. Moreover, thenoncanonical activation defect was specific to mutant B cells, because we did not find anydefect in LTβR-induced noncanonical p100 processing or RelB:p52 activation inNfkbiaNES/NES MEFs. Instead, our data indicate that the noncanonical activation defects inNfkbiaNES/NES B cells arose from the reduced transcripts for Nfkb2 and Relb genes andresulting reductions in their encoded noncanonical NF-κB proteins. Ferch et al. (2007)recently reported that B cell receptor (BCR) signaling induces Bcl10- and MALT1-dependent cRel activation whereas Bcl10, but not MALT1, is involved in RelA activation,thereby separating the mechanism of these canonical complex activation pathways. Inaddition, Stadanlick et al. (2008) and Castro et al. (2009) also reported that “tonic” BCR-stimulated NF-κB activity during B cell maturation promotes the de novo production ofcRel. They further showed that this cRel synthesis is required for sustained cRel activity andde novo p100 synthesis, which permitted BAFF (BLyS)-mediated noncanonical RelB:p52activation and B cell survival. Consistent with these reports, severe defects in constitutive(or tonic) and anti-IgM-induced cRel complex activity observed in mature NfkbiaNES/NES Bcells are associated with reduced expression of Rel and Nfkb2 genes and proteins. Moreover,we further found that expression of Relb mRNA and protein was also considerably reducedin mature NfkbiaNES/NES B cells. Transcription of Rel, Nfkb2, and Relb (as well as Nfkb1) iscontrolled by NF-κB in B cells (Castro et al., 2009; Grumont and Gerondakis, 1994;Perkins, 2007). Thus, nuclear export of IκBα is required for tonic (or constitutive) andsustained cRel activation, cRel-mediated synthesis of noncanonical NF-κB components,p100 and RelB, and the crosstalk between canonical and noncanonical NF-κB pathways inmature B cells. Defects in agonist-induced proliferation and apoptosis, expression of target

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genes associated with these processes, and B cell maturation defects at the T1–T2 transitionare all consistent with this model.

In contrast to the defects of B cell maturation, the abnormal formation of the spleen, inguinallymph nodes (LN), and Peyer’s patches (PP) observed in NfkbiaNES/NES mice have not beenreported in Rel−/− or Rel−/−Nfkb1−/− mice (Köntgen et al., 1995; Pohl et al., 2002). Theproper development of LN and PP requires the convergence of both canonical andnoncanonical NF-κB activation pathways through a combined action of lymphotoxin(LT)α1β2-LTβ receptor (R), tumor necrosis factor (TNF) α, or LTα3-TNFR1, and receptoractivator of nuclear factor kappa-B ligand (RANKL-RANK) pathways (Drayton et al., 2006;Hoffmann and Baltimore, 2006; Weih and Caamaño, 2003). In contrast to Nfkb1NES/NES

mice, Rankl−/− and Rank−/− mice show LN defects but the PP development and spleenarchitecture are largely normal (Dougall et al., 1999; Kong et al., 1999). Similarly, Tnfr1−/−

and Traf2−/− mice show PP defects but have normal LNs and spleen (Piao et al., 2007),again differing from NfkbiaNES/NES mice. Finally, Ltα−/− and Ltβr−/− mice lack all LN andPP and exhibit a disorganized spleen (Banks et al., 1995; De Togni et al., 1994) whereasLtβ−/− mice lack peripheral LN and PP but retain mesenteric and cervical LN (Koni et al.,1997). Ltα−/− and Ltβ−/− mice also show a reduction in serum IgA (Koni et al., 1997)similar to that seen in NfkbiaNES/NES mice, consistent with the role of mucosal lymphoidorgans, such as PPs, as the predominant producers of IgA (Kelsall, 2008). Clues to whyNfkbiaNES/NES mice have defective LN organogenesis confined to the inguinal LNs may liein the fact that development of LN groups requires distinct amounts of signaling ashighlighted by mice deficient in LTα, LTβ, and LTβR (Banks et al., 1995; De Togni et al.,1994; Koni et al., 1997). Thus, both canonical and noncanonical NF-κB functions in criticalcell types, including B cells, are probably sufficiently perturbed in NfkbiaNES/NES mice toinduce the observed secondary lymphoid organ and tissue defects that were not observed inRel−/− or Rel−/−Nfkb1−/− mice.

Unlike defects in B cells and secondary lymphoid organs and tissues, based on CD4 andCD8 cell surface staining alone we did not observe perturbations in CD4+ and CD8+ thymicT cell development or their numbers in thymus and spleen of the NfkbiaNES/NES mice. In Tcells, it is reported that IκBα is mostly associated with RelA, not cRel (Sen, 2006; Tam etal., 2001). RelA, but not cRel or p50, harbors an intrinsic NES motif (Harhaj and Sun, 1999;Tam et al., 2001). Accordingly, subcellular localization of inactive RelA complexes inNfkbiaNES/NES thymic and splenic T cells was mostly cytoplasmic even though a complexbetween RelA and mutant IκBα could be readily detected. Thus, it appears that the RelANES compensated for the lack of nuclear export function in NES mutant IκBα. However,consistent with recent reports demonstrating the role of cRel in the development ofregulatory T cells (Deenick et al., 2010; Hori, 2010; Isomura et al., 2009; Long et al., 2009;Ruan et al., 2009; Vang et al., 2010; Visekruna et al., 2010; Zheng et al., 2010), we foundevidence for developmental defects of this cell type in NfkbiaNES/NES mice. In addition,CD4+ but not CD8+ memory-like T cells in the mutant mice were also reduced, consistentwith previous reports on the role of cRel and p50 in the development of CD4+ memory-likeT cells (Zeng et al., 2008; Zheng et al., 2003). Therefore, molecular defects arising fromdefective IκBα export also impinge on these T cell subsets and more analyses are warrantedto fully investigate molecular, biological, and pathological consequences of reducedregulatory and memory-like T cells in NfkbiaNES/NES mice.

In conclusion, derivation and analysis of NfkbiaNES/NES mice revealed the surprisinglywidespread role for IκBα N-NES in vivo. To our knowledge, NfkbiaNES/NES mice representthe first in vivo model to directly evaluate the role of a specific NES motif in the regulationof the NF-κB-Rel family of transcription factors. Other mechanisms that control subcellulardistribution of NF-κB-Rel proteins, including RelA NES, IκBα C-NES, and IκBε NES,

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could also play important roles in a cell type- or context-dependent manner. Resolving invivo roles of these discrete mechanisms will necessitate creation and analysis of additionalanimal models. Similarly, although nucleocytoplasmic regulation mediated by specific NESmotifs has been documented for many other critical regulatory factors, including the tumorsuppressor p53 (Chu et al., 2007; Terry et al., 2007), direct examination of the functions ofmost of these NES-mediated export mechanisms in vivo remain to be performed. Additionalin vivo studies in which specific NES motifs are disrupted will help broaden ourunderstanding of the control of biological and pathological processes via active nuclearexport of regulatory factors.

EXPERIMENTAL PROCEDURESGeneration of NfkbiaNES/NES Mice

In brief, the targeting construct harboring a genomic Nfkbia locus with the triple IκBα N-NES mutation (M45A,L49A,I52A) introduced within exon 1 was electroporated into 129/SvR1 ES cells. Two correctly targeted ES cell clones (11D and 12G clones; Figure S1B) wereeach injected into C57BL/6J blastocysts to generate chimeras. Germline-transmittedNfkbia+/NES lines were then generated and subsequently crossed with EIIa-cre mice todelete the neo cassette in germline (Lakso et al., 1996), and the mice with mutant Nfkbiaallele lacking both the neo cassette and the EIIa-cre gene were back-crossed onto theC57BL/6J strain for 5–7 generations. Two independent Nfkbia+/NES lines were derived fromthe original two ES clones. The bulk of the data presented is derived from the analysis of11D mouse line. Similar results were also confirmed in a limited analysis of the 12G line.See Supplemental Experimental Procedures for further details.

Lymph Node and Peyer’s Patch AnalysesInguinal LNs were enumerated in situ by visual examination. LN volume was derived fromthe formula width2 × length/2 of histologic sections. PPs were enumerated by visualexamination of flushed small bowels. The aggregate volume of PPs in each mouse wasdetermined by the sum of estimated volumes for each PP as above.

Flow CytometrySingle-cell suspensions from BM, spleen, and lymph nodes were treated with Gey’s solutionto lyse red blood cells and then resuspended in phosphate-buffered saline (PBS) with 2%fetal bovine serum (FBS). The cells were stained with a combination of fluorescence-conjugated antibodies. Allophycocyanin (APC)-conjugated anti-B220 (17-0452), anti-IgM(17-5790-82), anti-CD4 (17-0042-82), fluorescein isothiocyanate (FITC)-conjugated anti-Thy1.2 (11-0902), anti-BAFFR (11-5943), phycoerythrin (PE)-conjugated anti-CD1d(12-0011), and anti-CD9 (12-0091) antibodies and PE-Cy5.5-conjugated streptavidin(35-4317) were purchased from eBioscience. PE-conjugated anti-B220 (553090), anti-CD43(553271), anti-CD21 (552957), anti-CD23 (553139), anti-CD5 (01035B), anti-CD8(553033), APC-conjugated anti-CD19 (550992), FITC-conjugated anti-CD21 (553818),anti-IgD (553439), and biotin-conjugated anti-CD23 (553137) were purchased from BDBiosciences PharMingen. All antibodies were mouse monoclonal antibodies. Apoptosis andcell cycle analyses were done by terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) and propidium iodide (PI) staining (Chen et al., 2008). Samples wereapplied to a flow cytometer (LSRII, Becton Dickinson). Data were collected and analyzedwith CellQuest software (Becton Dickinson).

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Bone Marrow TransplantationBone marrow (BM) cells were isolated from hind limbs of WT or NfkbiaNES/NES mice.Subsequently, cells (5 × 106) were injected retroorbitaly into sublethally irradiated (900rads) Jak3−/− recipients as previously reported (Chen et al., 2008). Eight weeks aftertransplantation, B cell development in the recipients was examined.

Immunocytochemical and Fluorescence AnalysesTissue sections were stained with tetramethylrhodamine isothiocyanate (TRITC)-conjugatedgoat anti-mouse IgM (Southern Biotechnology, Birmingham, AL), anti-B220 (BDBioscience PharMingen), and FITC-conjugated rat anti-mouse metallophilic macrophages,MOMA-1 (Serotec), as previously reported (Chen et al., 2008). Images were taken withfluorescence microscope (Zeiss Axioskop, Carl Zeiss Inc., Jena, Germany) with a 10×objective lens (numerical aperture 0.3) and a charge-coupled device (CCD) camera (Sensys,Photometrics, Tucson, AZ). Alternatively, images were acquired with an Olympus BX41microscope (Olympus America) and an Olympus DP20 camera, and data were analyzedwith the MetaMorph Version 6.1 software (Molecular Devices, Downington, PA).

Analysis of NF-κB and IκBα Subcellular LocalizationSplenic B cells were purified by AutoMACS depletion with anti-CD4−, anti-CD8−, andanti-Mac-1-conjugated microbeads (Miltenyi Biotec). The purified cell populations wereseeded onto CC2-treated four-well glass chamber slides (Lab-Tek). Cells were stained as inO’Connor et al. (2005). Each experiment was repeated at least twice and 200 cell countswere conducted in triplicate to yield % ± SD.

EMSA, Immunoblotting, and Immunoprecipitation AnalysesSplenic B cells were isolated by negative selection as above. Immature (AA4.1+) and mature(AA4.1−) B cells were separated with biotin-conjugated anti-AA4.1 (eBioscience) and anti-streptavidin microbeads (Miltenyi Biotec). EMSA, immunoblot, and immunoprecipitationanalyses were performed as previously described (O’Connor et al., 2004). The antibodiesused for immunoblotting were anti-RelA (C-20), anti-RelB (C-19), anti-p52 (C-5), anti-p50(E-10), anti-IκBα (C-21), anti-IκBβ (C-20), and anti-Actin (I-19) purchased from Santa CruzBiotechnologies. The cRel antibody (5071) was previously described (Inoue et al., 1991).Anti-tubulin (DM1A) was purchased from Calbiochem.

Proliferation AssayCell proliferation was analyzed by 3H-thymidine incorporation assay as previously reported(Chen et al., 2008).

Quantitative RT-PCR and NF-κB Signaling Pathway PCR Array AnalysisSee Supplemental Experimental Procedures for details.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank A. Griep, P. Powers, C. Bartley, and M. Lye at the Transgenic Animal Facility at the University ofWisconsin Biotechnology Center for the gift of the TK-pflox8 targeting vector and assistance with the generation ofthe mutant mice, B. Seufzer for genotyping of the mice, and members of the S.M., D.W., and R.W. labs forstimulating discussions. This work was supported in part by National Institutes of Health grants R01 CA081065,R56 CA081065, CA077474, and R01 GM083681 (S.M.), T32 CA009614 (D.Y.), R01 AI083453 and P01

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GM071862 (A.H.), R01 AI52327 (R.W.), and R01 HL073284, R01 AI079087, and P01 HL44612 (D.W.), byUWCCC Core grant P30 CA14520 (G. Wilding, PI), and by Scholar Award from the Leukemia & LymphomaSociety (D.W.).

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Figure 1. Defects of Proper Formation of Spleen, Inguinal LNs, and PPs in NfkbiaNES/NES Mice(A) Photographs of inguinal LNs in situ from WT and NfkbiaNES/NES littermates (top). LNswere counted in WT (n = 35) and NfkbiaNES/NES (n = 35) mice and displayed graphically(bottom).(B) Inguinal LNs in WT and NfkbiaNES/NES mice were analyzed by hematoxylin and eosin(H&E) stain and immunostaining with anti-B220.(C) Inguinal LN volume was derived by the formula width2 × length/2 of histologic sectionsfrom wild-type (n = 32) and NfkbiaNES/NES (n = 11) mice and displayed graphically. Thedifference is statistically significant (*p < 0.001, unpaired t test).

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(D) PPs were counted from WT (n = 6) and NfkbiaNES/NES (n = 7) mice and displayedgraphically.(E) Aggregate size of PPs were calculated as in (C) from samples in (D). Data are mean ±SD, *p < 0.001 versus WT.(F) Spleens from WT and NfkbiaNES/NES mice were analyzed by H&E stain andimmunostaining with anti-B220, 4× objective.(G) Immunofluorescent histochemical analysis of the spleens from WT or NfkbiaNES/NES

mice were analyzed with anti-MOMA-1 and anti-IgM. MZ B cell layer is external to thering of metallophilic macrophages.(H) Thymii from WT or NfkbiaNES/NES mice were analyzed by H&E stain, 4× objective.

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Figure 2. Severely Impaired B Cell Maturation in NfkbiaNES/NES MiceComparisons between WT and NfkbiaNES/NES mice are shown in each panel.(A) Splenocytes were stained with anti-B220 and anti-Thy1.2. Percentages above each boxindicate cells in the gated lymphoid population.(B) The numbers of total splenocytes and total splenic B and T cells are displayedgraphically.(C) Thymocytes from WT and NfkbiaNES/NES mice were stained with anti-CD4 and anti-CD8. Percentages indicate cells in the gated lymphoid population (C and E–G).(D) The numbers of total thymocytes and DN, DP, CD4+, and CD8+ T cells in the thymus ofWT and NfkbiaNES/NES mice are displayed graphically.

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(E) Splenocytes were stained with anti-B220, anti-IgM, and anti-IgD. For cells gated onB220+, T1 (IgMhiIgD−), T2 (IgMhiIgD+), and FO (IgMloIgD+) B cells are shown.(F) Splenocytes were stained with anti-IgM, anti-CD21, and anti-CD23. For cells gated onCD23+, T2 (CD21hiIgMhi) and FO (CD21intIgMlo) B cells are shown. For cells gated onCD23−, T1 (CD21loIgMhi) and MZ (CD21hiIgMhi) B cells are shown.(G) Splenocytes were stained with anti-B220, anti-CD21, and anti-CD23. For cells gated onB220+, MZ B cells (CD21hiCD23lo) are shown.(H) Top: Splenocytes were stained with anti-B220, anti-CD1d, and anti-CD9. For cells gatedon B220+, MZ (CD1d+CD9+) cells are shown. Bottom: Splenocytes were stained with anti-IgM, anti-CD1d, and anti-CD23. For gated lymphoid cells, T2 (CD1d+CD23+) and MZ(CD1d+CD23−) cells are shown.(I) The numbers of T1, T2, FO, and MZ B cells obtained from (F) are displayed graphically.(J) Serum Ig amounts were determined by ELISA. The mean value and standard deviation ofthe serum Ig amounts were calculated.Data shown are representative of or obtained from 7 (A–G, I), 2 (H), or 10 (J) mice of eachgenotype.

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Figure 3. Severely Impaired B Cell Maturation in Recipients of BM from NfkbiaNES/NES MiceSublethally irradiated Jak3−/− mice were transplanted with BM from WT (Jak3−/− +Nfkbia+/+) or NfkbiaNES/NES (Jak3−/− + NfkbiaNES/NES) mice. Irradiated Jak3−/− micewithout BM transplantation were used as a negative control.(A) BM from the recipient mice were stained with anti-B220 and anti-IgM. Percentages hereand below indicate cells in the gated lymphoid population.(B) Splenocytes from the recipients were stained with anti-B220 and anti-Thy1.2.(C) Splenocytes from the recipients were stained with anti-IgM, anti-CD21, and anti-CD23.In cells gated on CD23+, T2 and FO B cells are shown. In cells gated on CD23−, T1 and MZB cells are shown.

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(D) Splenocytes from the recipients were stained with anti-B220, anti-CD21, and anti-CD23. In cells gated on B220+, MZ B cells are shown.Data are representative of five recipients transplanted with each genotype of BM. Controlsin (C) and (D) are not shown due to very low percentages.

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Figure 4. Defects in NF-κB Activation and cRel and IκBα Localization in NfkbiaNES/NES B Cells(A) Total cell lysates of unstimulated splenic AA4.1− mature B cells from WT andNfkbiaNES/NES mice were analyzed by a supershift assay to detect different NF-κB familymembers.(B and C) Splenic AA4.1− mature B cells of WT and NfkbiaNES/NES mice were stimulatedwith anti-IgM (10 µg/mL, B) and LPS (10 µg/mL, C) for the indicated times. Total cellextracts were made and NF-κB activity was measured by EMSA with an Igκ-κB probe.Variable increases in p50 homodimer binding seen in (B) are probably due to enhanceddetection of p50 homodimer that has a lower affinity to the Igκ-κB site used because of thelack of competition by high-affinity heterodimer complexes for a limited amount of probe

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used. A short exposure time is shown in (C) to highlight the difference of induced activity(the basal activity difference is thus less evident because of underexposure).(D and E) Samples in (B) and (C) were analyzed by immunoblotting with anti-IκBα, -IκBβ,and -actin.(F) The IKK complex was immunoprecipitated from B cells stimulated with LPS (10 µg/mLfor 30 min) and an IKK kinase assay was performed with GST-IκBα(1–66aa) as substrate.(G) Splenic AA4.1− mature B cells were fixed and stained with anti-IκBα and anti-cRel andcounterstained with Hoechst dye.(H) Percentages ± 1 SD of pronounced nuclear staining for each protein as in (G) werederived from triplicate counts of random 200 cells. **paired t test determined a statisticallysignificant difference p < 0.001.(I) Quantitation of nuclear and cytoplasmic IκBα and cRel in splenic AA4.1− mature B cells.Images of stained cells as in (G) were analyzed by Image J software. Mean intensity ofstaining in the nuclear and cytoplasmic compartments of 10 random cells stained with eachantibody, normalized to background intensity, and expressed as a ratio of mean nuclearintensity/mean cytoplasmic intensity (N/C) are shown as columns with 1SD bars. Paired ttest determined a statistically significant difference in N/C ratio of IκBα and cRel betweenWT and mutant cells, **p < 0.001.(J) Lysates obtained from splenic AA4.1− mature B cells of WT and NfkbiaNES/NES micewere incubated with anti-cRel and the precipitates were analyzed by immunoblotting withanti-cRel, -IκBα, and -IκBβ (left). Inputs were blotted with the same antibodies and an anti-tubulin loading control (right).

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Figure 5. Reduced Expression of Noncanonical NF-κB Members and Activation inNfkbiaNES/NES B Cells(A) Splenic AA4.1− mature B cells were purified from Nfkbia+/+, Nfkbia+/NES, andNfkbiaNES/NES mice and total cell extracts were made. The expression of NF-κB and IκBfamily members were analyzed by immunoblotting.(B) Total RNA from splenic AA4.1− mature B cells of WT and NfkbiaNES/NES mice wereanalyzed by an RT2 Profiler Mouse NF-κB Signaling Pathway PCR Array (SA Biosciences)and the statistical analysis was done according to the manufacturer’s instructions.(C) Splenic AA4.1− mature B cells of WT and NfkbiaNES/NES mice were treated withcycloheximide (25 µg/ml) for indicated times or with LPS alone (10 µg/ml, 1 hr) and totalcell lysates were analyzed for IκBα and IκBβ degradation by immunoblotting. The relativedegradation as measured by laser scanning of each band followed by NIH ImageJ analysisnormalized to actin loading control showed that the rate of mutant IκBα degradation was 2-fold slower than that of the WT protein.(D) Splenic AA4.1− mature B cells purified from WT and NfkbiaNES/NES mice were treatedwith BAFF (500 ng/mL) for 24 hr. The processing of p100 to p52 was detected byimmunoblot with an anti-p52.

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(E) EMSA analysis was performed with samples in (D) via an Igκ-κB probe.(F) MEF cells generated from NfkbiaNES/NES and WT mice were treated with anti-LTβR (0.5µg/ml) for the indicated times (hr). The processing of p100 was detected by immunoblotwith anti-p52 along with antibodies to other NF-κB and IκB family members.(G) EMSA analysis of extracts from (F) was performed as in (E).

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Figure 6. Defective Proliferation and Survival of NfkbiaNES/NES B Cells(A and B) Splenic immature (AA4.1+) and mature (AA4.1−) B cells purified from WT andNfkbiaNES/NES mice were stimulated with anti-IgM with or without IL-4 or with LPS.Proliferation was assessed by [3H]thymidine incorporation. Data are representative of atleast four independent experiments.(C and D) Splenic immature (AA4.1+) and mature (AA4.1−) B cells were purified from WTand NfkbiaNES/NES mice and then stimulated with anti-IgM plus IL-4 or with LPS.Subsequently, cells were collected, stained with propidium iodide, and analyzed for cellcycle profile by flow cytometry. The percentages of cells in subG0, G0–G1, and S-G2-M areindicated.

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(E and F) Splenic mature (AA4.1−) B cells purified from WT and NfkbiaNES/NES mice werestimulated with IgM antibody or with LPS. RNA expression amounts of Bcl2l1 (encodingBcl-XL) and Myc were determined with real-time reverse transcription PCR. Data were donein triplicate from three and two mice each for Bcl2l1 and Myc, respectively.(G) Spenic and thymic T cells purified from WT and NfkbiaNES/NES mice were stimulatedwith indicated agonists and analyzed as in (A).

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Table 1

Summary of Defects Seen in Nfkbia+/NES and NfkbiaNES/NES Mice

Phenotypes Nfkbia+/+ Nfkbia+/NES NfkbiaNES/NES

B Cells (×106)

Bone marrow (6)

Total B 9.3 ± 1.1 12.1 ± 2.5 11.7 ± 2.3

Pro pre-B 4.2 ± 0.7 6.8 ± 1.5** 7.3 ± 1.3**

Immature B 1.3 ± 0.2 2.1 ± 1.0 2.0 ± 0.8

Mature B 2.6 ± 0.4 2.0 ± 0.5 1.3 ± 0.4**

Spleen (7)

Total B 26.6 ± 7.1 14.9 ± 2.6** 12.9 ± 3.3**

Transitional T1 1.3 ± 1.5 1.3 ± 0.7 1.5 ± 1.1

Transitional T2 5.7 ± 2.0 1.9 ± 1.1** 0.8 ± 0.7**

Follicular 12.4 ± 3.4 7.8 ± 2.0** 4.7 ± 1.3**

Marginal zone 0.7 ± 0.2 0.3 ± 0.2** 0.2 ± 0.1**

Lymph nodes (4)

Total B 1.0 ± 0.5 0.3 ± 0.1* 0.2 ± 0.1*

T Cells (×106)

Thymus (7)

Total T 95.6 ± 30.1 84.7 ± 26.0 64.6 ± 26.3

CD4−CD8− 2.0 ± 0.8 1.5 ± 0.4 1.3 ± 0.6

CD4+CD8+ 83.7 ± 25.9 73.6 ± 23.6 54.8 ± 21.9

CD4+CD8− 6.7 ± 2.5 6.7 ± 1.6 6.0 ± 2.7

CD4−CD8+ 2.8 ± 1.1 2.7 ± 0.9 2.2 ± 1.2

Spleen (7)

Total T 19.6 ± 4.4 18.5 ± 2.3 23.3 ± 4.9

CD4+CD8− 12.0 ± 2.9 11.8 ± 1.6 14.7 ± 3.4

CD4−CD8+ 7.5 ± 1.7 6.7 ± 0.9 8.5 ± 1.8

Lymph nodes (4)

Total T 3.7 ± 1.2 1.9 ± 0.2* 1.9 ± 1.0

CD4+CD8− 2.2 ± 0.7 1.2 ± 0.1* 1.2 ± 0.6

CD4−CD8+ 1.5 ± 0.5 0.7 ± 0.1* 0.7 ± 0.4*

Inguinal LNs (16)

Mean number 2.0 1.8 0.7**

Mean volume 3.4 ± 1.7 2.0 ± 0.9* 1.2 ± 0.6**

Peyer’s Patches (5–7)

Mean number 6.5 6.4 1.7**

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Phenotypes Nfkbia+/+ Nfkbia+/NES NfkbiaNES/NES

Aggregate volume (mm3) 30.3 ± 8.8 17.8 ± 4.3** 2.3 ± 1.7**

Numbers in parentheses refer to the number of mice analyzed for the values shown.

Significance at *p < 0.05 or **p < 0.01.

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