A Novel Mutation in the Nfkb2 Generates an NF- κB2 ''Super Repressor'' Elena Tucker, Kristy O'Donnell, Martina Fuchsberger, Adrienne A. Hilton, Donald Metcalf, Kylie Greig, Natalie A. This information is current as Sims, Julian M. Quinn, Warren S. Alexander, Douglas J. of September 23, 2021. Hilton, Benjamin T. Kile, David M. Tarlinton and Robyn Starr J Immunol 2007; 179:7514-7522; ;

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel Mutation in the Nfkb2 Gene Generates an NF-␬B2 “Super Repressor”1

Elena Tucker,*‡ Kristy O’Donnell,† Martina Fuchsberger,* Adrienne A. Hilton,† Donald Metcalf,† Kylie Greig,† Natalie A. Sims,*‡ Julian M. Quinn,*‡ Warren S. Alexander,†§ Douglas J. Hilton,†§ Benjamin T. Kile,†§ David M. Tarlinton,†§ and Robyn Starr2*‡

The noncanonical NF-␬B pathway regulates the development and function of multiple organs and cell lineages. We have generated mice harboring a novel mutation in Nfkb2 that prevents the processing of the inhibitory precursor, p100, into the active subunit, p52. Mutant mice express a complex phenotype with abnormalities in a variety of tissues, and with a spectrum that is more severe than in mice carrying a targeted deletion of Nfkb2. Signaling through the noncanonical pathway is ablated due to the absence of p52, resulting in disorganized splenic architecture and disrupted B cell development. The inhibitory precursor form of NF-␬B2 interacts with RelA, preventing activation of RelA dimers in response to both canonical and noncanonical stimuli, which in Downloaded from combination with p52 deficiency, results in defective lymph node formation and bone homeostasis. These findings demonstrate a key role for NF-␬B2 in the regulation of RelA activation and suggest overlap in the function of NF-␬B members in canonical and noncanonical pathway signaling. The Journal of Immunology, 2007, 179: 7514–7522.

he NF-␬B transcription factors are critical regulators of (IKK)␣ (5). IKK␣ phosphorylates p100 at serines 866 and 870 at the development and function of the immune system. the C terminus, enabling recruitment of the E3 ligase ␤-transducin http://www.jimmunol.org/ T Mammalian cells contain five NF-␬B members (RelA, repeat-containing (␤TrCP) and partial processing by the RelB, c-Rel, NF-␬B1, and NF-␬B2), each of which has an N- proteasome to yield p52 (6). terminal with sequences for dimerization, The “canonical” NF-␬B pathway has a central role in the in- DNA binding, and nuclear localization (1). In resting cells, NF-␬B flammatory response. This pathway predominantly activates p50: dimers are maintained in an inactive state by interaction with I␬B RelA dimers and is triggered by a variety of proinflammatory li- , which mask the nuclear localization of NF-␬B proteins, gands, including TNF-␣, LPS, and viral proteins (2). By contrast, thereby preventing their nuclear migration. Activation of the a limited number of TNF family cytokines activate the “nonca- ␬ ␬ NF- B pathway by cytokines and other ligands leads to proteolytic nonical” NF- B pathway; lymphotoxin (LT), activator of by guest on September 23, 2021 processing of I␬B proteins, allowing NF-␬B dimers to translocate NF-␬B ligand (RANKL), CD40L, and B cell activating factor (7). to the nucleus and activate the transcription of target (1). Although the noncanonical pathway mostly activates p52:RelB NF-␬B1/p50 and NF-␬B2/p52 are synthesized as precursor pro- dimers, both RelA and RelB containing NF-␬B dimers are acti- teins p105 and p100, respectively. The C-terminal regions of p105 vated by these stimuli (8). The noncanonical pathway regulates the and p100, containing domains, function as I␬B and development and organization of secondary lymphoid organs (via ␬ retain NF- B proteins in the cytoplasm (2). Processing of the pre- LT signaling), medullary thymic epithelial cell differentiation cursors into their active forms p50 and p52 is required for nuclear (RANKL), thymocyte emigration (LT), B lymphocyte survival and migration (1). In contrast to p105, which undergoes constitutive maturation (CD40L and B cell activating factor), and bone ho- processing (3), p100 processing is tightly regulated and is triggered meostasis (RANKL) (9–14). by external ligands (4). Activation by ligand increases the stability Mice harboring mutations that affect NF-␬B2 expression or ␬ 3 ␬ of NF- B-inducing kinase (NIK), which recruits I B kinase function have revealed a complex range of physiological roles for this molecule. Nfkb2Ϫ/Ϫ mice, which lack both p100 and p52, have *St. Vincent’s Institute, Fitzroy, Victoria, Australia, †The Walter and Eliza Hall disorganized splenic architecture, small or absent peripheral lymph Institute, and ‡Department of Medicine, and §Department of Medical Biology, node (LN), and a reduced number of mature B cells (9, 10, 15–17). University of Melbourne, Parkville, Victoria, Australia Mice engineered to process p100 constitutively display abnormal Received for publication September 25, 2007. Accepted for publication September lymphocyte proliferation and enlarged spleen and LN, and die dur- 25, 2007. ing the perinatal period (18). Similar defects leading to unregulated The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance p52 production are associated with the development of lymphoma with 18 U.S.C. Section 1734 solely to indicate this fact. in humans (19, 20). Collectively, these studies indicate that p52 1 This work was supported by Program Grant 257500 from the National Health and levels are strictly controlled, and loss of this regulation results in Medical Research Council. R.S. is supported by a Viertel Senior Medical Research severe immune abnormalities. Fellowship. NIK plays a pivotal role in p100 phosphorylation and process- 2 Address correspondence and reprint requests to Dr. Robyn Starr, St. Vincent’s In- Ϫ/Ϫ stitute, 41 Victoria Parade, Fitzroy Victoria 3065, Australia. E-mail address: ing. The phenotype of Nik mice or alymphoplasia (aly) mice, Ϫ Ϫ [email protected] which have a mutant form of NIK, is similar to that of Nfkb2 / 3 Abbreviations used in this paper: NIK, NF-␬B-inducing kinase; IKK, I␬B kinase; mice, but the mesenteric LNs are absent in addition to peripheral ENU, N-ethyl-N-nitrosourea; LT, lymphotoxin; LN, lymph node; RANKL, receptor LNs (21, 22). In this respect, these mice more closely resemble activator of NF-␬B ligand; SP, single positive; wt, wild type. mice in which both the canonical and noncanonical NF-␬B path- Ϫ Ϫ Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 ways are compromised, including LT␣-deficient mice (Lta / )

www.jimmunol.org The Journal of Immunology 7515

FIGURE 1. Lym1 is a nonsense mutation in Nfkb2. A, Lymphocytosis in affected members of the Lym1 pedigree. B, Absence of inguinal LN (indicated by arrow) in a Lym1/ϩ mouse. C, Mapping haplotypes for Lym1. Markers used are indicated, and defining recombination events are shaded gray. Filled squares indicate C57BL/6 homozygosity and divided squares indicate C57BL/6:BALB/c heterozygosity. The candidate region is between D19Mit53 and D19Mit49. D, T to A transversion at Nfkb2 base 2854 in genomic DNA of a Lym1/ϩ mouse. E, Sche- Downloaded from matic representation of the domain structure of NF-␬B2. The Lym1 mutation is predicted to re- place the codon that encodes aa 868 with a pre- mature stop codon. Residues critical for process- ing are highlighted in bold. ARD, ankyrin repeat domain; CPS, C-terminal phosphorylation site;

GRR, glycine-rich region; K, lysine; NPS, N-ter- http://www.jimmunol.org/ minal phosphorylation site; RHD, Rel homology domain. F, Extracts from wt and Nfkb2Lym1/ϩ mouse embryonic fibroblasts were blotted with an NF-␬B2-specific Ab, showing the reduced size of p100Lym1 in Nfkb2Lym1/ϩ cells. G, Geno- typing strategy for the Lym1 mutation. by guest on September 23, 2021

(23), LT␤ receptor-deficient mice (LtbrϪ/Ϫ) (11), and mice lacking RelA, leading to more severe defects than those found in Nfkb2Ϫ/Ϫ both NF-␬B1 and NF-␬B2 (17). Like Nfkb2Ϫ/Ϫ mice, Nfkb1Ϫ/Ϫ mice. We provide evidence in vivo that NF-␬B2 function is not mice have partial defects in LN formation, lacking inguinal LNs limited to the regulation of p52:RelB activation, and that a key but with the mesenteric LNs retained (17). The more severe LN function of p100 is to interact with and regulate RelA-containing defect seen when both pathways are perturbed suggests that mul- complexes. tiple NF-␬B molecules participate in the development of LN and to some extent can compensate for the loss of the other. Therefore, the complete absence of LN in NikϪ/Ϫ mice, which retain p100 but Materials and Methods Generation and screening of mutant mice lack p52, raises the possibility that the I␬B function of p100 may not be restricted to regulating p52:RelB activity, but may also Male BALB/c mice were treated with N-ethyl-N-nitrosourea (ENU) as de- serve to regulate other NF-␬B molecules. scribed (27). ENU-treated mice were mated with isogenic females to yield G progeny, which were bled at 7 wk of age, and the number of PBLs Several recent studies have provided additional evidence for 1 ␬ determined using an Advia 120 Automated Hematological Analyzer cross-talk between the canonical and noncanonical NF- B path- (Bayer). Mice were routinely housed in clean, conventional facilities and ways. In a study of osteoclastogenesis in NIK-deficient mice, p100 were used for experiments at 8–14 wk of age. All experiments with mice was shown to bind p50:RelA complexes and prevent their nuclear were approved by St. Vincent’s Hospital Animal Ethics Committee. translocation (24). Using genetic mutants lacking the three canon- ical I␬B proteins, p100 was shown to interact with and regulate Genetic mapping p50:RelA complexes in response to noncanonical stimuli (25). Mice heterozygous for the Lym1 mutation on a BALB/c background were

Similarly, in naive T cells p100 forms complexes with p50:RelA to mated with C57BL/6 wild-type (wt) mice. F1 mice were identified at 7 wk regulate T cell activation (26). of age by elevated PBL counts and intercrossed to produce F2 mice. DNA In this study, we describe the characterization of mice harboring was prepared from the liver of F2 mice and simple sequence length poly- morphisms spaced evenly throughout the genome were amplified and a novel mutation in Nfkb2. This mutant allele encodes a nonproc- analyzed. The Lym1 mutation was localized to 19, and the essible form of p100, preventing p52 formation. Furthermore, the candidate interval was refined via analysis of additional Mit marker and inhibitory precursor form of NF-␬B2 inhibits the activation of in-house CA repeat marker in the region. 7516 A NONPROCESSIBLE MUTANT FORM OF NF-␬B2

FIGURE 2. Abnormal splenic ar- chitecture and inflammation in Nfkb2Lym1/Lym1 mice. Spleen sections (A–D) from wt and Nfkb2Lym1/Lym1 mice were stained for expression of B220 (brown) and CD3 (blue) (A), CD35 (brown) and CD3 (blue) (B), IgM (brown) and IgD (blue) (C), and MOMA1 (D). Lung sections (E–H) from wt and Nfkb2Lym1/Lym1 mice show focal inflammatory cell infiltra- tion (H&E stain) (E) containing T cells (anti-CD3) (F), B cells (anti- CD45R) (G), and macrophages (F4/80 Ab) (H). Original magnifica- tion ϫ20 (lung) and ϫ40 (spleen). Data are representative of two to three Downloaded from independent experiments. I, Aire ex- pression in total thymocytes was mea- sured by real-time quantitative PCR. Results are expressed relative to a wt thymus sample and represent mean ϩ SD of triplicate measurements from http://www.jimmunol.org/ three to four mice of each genotype. p Ͻ 0.01. J, Increased mortality of ,ءء Nfkb2Lym1/Lym1 mice. The proportion of wt (n ϭ 20), Nfkb2Lym1/ϩ (n ϭ 20), and Nfkb2Lym1/Lym1 (n ϭ 10) mice re- maining disease-free (percent healthy) with age is shown. by guest on September 23, 2021

Sequencing and genotyping bers, 2 ϫ 104 Calibrite beads (BD Biosciences) were added to each well before harvest. DNA was prepared from tail biopsy and each exon of Nfkb2 was PCR- amplified and sequenced on an automated sequencer (Applied Biosystems). Measurement of serum Ig Genotyping was performed by PCR amplification of exon 23 of Nfkb2, followed by digestion with Bsu 36I (New England Biolabs). In the pres- Serum Abs were measured using isotype-specific ELISA, as described (28). ence of the Lym1 mutation, a wt fragment (233 bp) is digested into two Briefly, serum Abs were captured with goat anti-mouse isotype-specific sera smaller fragments of 144 and 89 bp. (Southern Biotechnology Associates) and revealed with isotype-specific, HRP- conjugated goat anti-mouse sera (Southern Biotechnology Associates). Puri- Real-time quantitative PCR fied myeloma proteins were used as controls. Total RNA was isolated from thymocyte suspensions using TRIzol reagent (Invitrogen Life Technologies). Real-time quantitative PCR analysis was Histology and immunohistochemistry performed with primer and probe Assay-on-demand sets (Applied Biosys- Frozen spleen sections were stained with unlabeled anti-B220 (clone RA3- ␤ tems) for Aire and -actin as a housekeeping reference gene. Analysis was 6B2), anti-CR2/CR1 (clone 7G6; BD Biosciences) or anti-IgD (clone 11- performed on a Rotor-Gene RG-3000 cycler (Corbett Research). Results rep- 26c) Abs, or biotinylated anti-CD3 (clone KT3), IgM (clone 331.12), and ϩ resent mean SD of triplicates from three to four mice of each genotype. MOMA-1 Abs. Unlabeled Abs were detected with a monoclonal anti-rat Ab conjugated to HRP (clone G16-510E3; BD Biosciences) and biotinyl- Abs and flow cytometry ated Abs with streptavidin-alkaline phosphatase (Southern Biotechnology Single cell suspensions were stained as described (28) with Abs specific for Associates). Alkaline phosphatase was visualized with the Fast Blue kit CD23 (clone B3B4), CD21 (7G6), B220 (RA3-6B2), CD4 (GK1.5), CD8 (Vector Laboratories), with endogenous phosphatases blocked by the ad- (53-6.7), CD69 (H1.2F3) and CD62L (Mel-14), and analyzed on a LSR, dition of 2 mM levamisole (Sigma-Aldrich). HRP was visualized with the FACSCalibur, or FACSAria flow cytometer (BD Biosciences). 3-amino-9-ethylcarbazole substrate kit (Vector Laboratories). For lung sec- tions, organs were fixed in 4% paraformaldehyde, embedded in paraffin, B cell purification and culture sectioned and stained with H&E. Immunohistochemistry was performed as described, using Abs specific for CD45R, CD3, or F4/80 (Serotec) (29). Small, resting B cells were isolated from spleen suspensions using a com- bination of Percoll gradient centrifugation and anti-B220 MACS beads Immunoblotting and immunoprecipitation (Miltenyi Biotec). B cells were stimulated for 3 days in RPMI 1640 with 5% FCS and 10Ϫ4 M 2-ME containing either an optimal dilution of bac- B cells purified from mouse spleen using anti-B220 MACS beads (5 ϫ ␮ ␮ Ј 6 ulovirus-derived CD40L, 20 g/ml LPS (Difco) or 10 g/ml F(ab )2 goat 10 /sample) were either untreated or stimulated for4hat37°C with 10 anti-mouse ␮ chain (Jackson ImmunoResearch Laboratories). Viability of ␮g/ml anti-CD40 Ab (clone FGK45). Cytoplasmic and nuclear fractions cells cultured in medium alone was determined by flow cytometry at 24-h were prepared using the Qproteome cell compartment kit (Qiagen). Mac- intervals by propidium iodide exclusion. For quantification of cell num- rophages were derived from the bone marrow of mice by culture in L The Journal of Immunology 7517

FIGURE 3. Perturbed T cell develop- ment in Nfkb2Lym1/Lym1 mice. Thymo- cytes (A), PBLs (B), and splenocytes (C) from Nfkb2Lym1/Lym1 mice (L/L) and wt (ϩ/ϩ) controls were stained with the in- dicated Abs and analyzed by flow cy- tometry. CD69 vs CD62L profiles were Downloaded from gated on CD4 SP cells. The percentage µ of cells in each quadrant is indicated. Total cell number and the number of in- dividual cell subsets for each organ are quantified on the right. Results are ex- pressed as mean ϩ SD of cell counts from five mice (8-wk-old) of each geno- type. C, Spleen weight of five mice of http://www.jimmunol.org/ each genotype is indicated in a scatter- plot (bottom right). Horizontal bar indi- cates mean weight. DP, double positive; p Ͻ ,ءء ;p Ͻ 0.05 ,ء .DN, double negative .p Ͻ 0.001 ,ءءء ;0.01 by guest on September 23, 2021

cell-conditioned medium for 7 days. Bone marrow macrophages were stim- acid phosphatase-positive cells with three or more nuclei were counted as ulated for 3 h with 1 ␮g/ml LPS followed by elution with Cell Dissociation osteoclasts. Histomorphometric analysis was conducted on L4 lumbar ver- buffer (Invitrogen Life Technologies). Lysates from equivalent numbers of tebrae as previously described (33). cells were resolved by SDS-PAGE and immunoblotted using Abs specific for RelA (F-6), NF-␬B1 (E10), or NF-␬B2 (C5) (Santa Cruz Biotechnol- Statistical analyses ogy), Apaf-1 (cytoplasmic) a gift from Dr. D. Huang (Walter and Eliza t Hall Institute (Melbourne, Australia), and poly(ADP-ribose) polymerase Significant differences were determined by Student’s two-tailed tests for (nuclear) (clone 42; BD Biosciences). Mouse embryonic fibroblast cell independent events or one-way ANOVA followed (where significant) by lines were generated from E13 embryos by trypsin dissociation of tissue, as Fisher’s protected least significant difference post hoc test to identify the B–D described (30). For immunoprecipitation, mouse embryonic fibroblasts significant pairwise differences (see Fig. 5, ). were stimulated for 15 h with 1 ␮g/ml agonist LT␤R-specific Ab (AF.H6; BD Biosciences) and whole cell lysates (1 ϫ 106/sample) were prepared in Results lysis buffer (20% glycerol, 0.2 mM EDTA, 0.5% Nonidet P-40, 150 mM Generation of the Lym1 pedigree NaCl) and immunoprecipitated overnight at 4°C with 2 ␮g of anti-RelA Ab (F-6). SDS-PAGE and Western blotting were performed essentially as de- To identify genes important for immune regulation, we conducted scribed (31). Band intensity on films was quantitated using Scion Image a forward genetic screen using the chemical mutagen ENU. A software. cohort of mutant mice was generated by crossing male ENU- Osteoclast differentiation assays and histomorphometry treated BALB/c mice with BALB/c females. The resultant G1 progeny were bled at 7 wk of age, and the different hemopoietic The osteoclastogenic potential of murine bone marrow was determined in cell subsets were quantified using an automated blood analyzer. vitro as previously described (32). Briefly, 1 ϫ 105 bone marrow cells were incubated with 100 ng/ml RANKL and 25 ng/ml M-CSF, with change of A pedigree of mice (Lym1) was found in which approximately medium and mediators at day 3. After 7 days, cells were fixed and stained half the members exhibited an increased number of circulating histochemically for tartrate-resistant acid phosphatase. Tartrate-resistant lymphocytes (Fig. 1A). By mating affected Lym1 mice to BALB/c 7518 A NONPROCESSIBLE MUTANT FORM OF NF-␬B2

FIGURE 4. Perturbed B cell maturation and function in Nfkb2Lym1 mice. A, Normal B cell de- velopment in the bone marrow of Nfkb2Lym1/Lym1 mice. Bone marrow from Nfkb2Lym1/Lym1 mice (L/L) and wt (ϩ/ϩ) controls was stained with the indicated Abs and analyzed by flow cytometry. The percentage of pro- and pre-B cells (B220low IgMϪ), immature B cells (B220low IgMϩ), and recirculating mature B cells (B220high IgMϩ)is indicated. Cell count of each subset is quantified on the right. B, Splenic B cell subsets. Spleno-

cytes were stained with the indicated Abs. Dot Downloaded from plots gated on CD23Ϫ and CD23ϩ populations show the proportions of marginal zone (MZ) B cells and transitional stage 1 (T1) B cells (mid- dle) and transitional stage 2 (T2) and follicular B cells (lower). Cell counts of each subset are graphed below. Data in A and B show mean ϩ

SD of cell counts from three to five mice (8-wk- http://www.jimmunol.org/ old) of each genotype. C, Serum Ig levels in na- ive mice. Horizontal bar indicates mean level. Wt (ϩ/ϩ) mice (E) and Nfkb2Lym1/Lym1 mice (F) are shown. D, Purified B cells were cultured for 3 days in the indicated stimuli, and total live cells displayed. E, Viability of purified B cells cul- tured in medium alone was determined by flow cytometry at 24-h intervals by propidium iodide (PI) exclusion. Data in D and E represent µ mean Ϯ SD of triplicate measurements, and are by guest on September 23, 2021 representative of two to three independent exper- p Ͻ ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .iments 0.001.

wt mice, we confirmed that the lymphocytosis was inherited as a Sequence analysis of the 23 exons of Nfkb2 revealed a thymine dominant trait (data not shown). Lym1 heterozygous mice were to adenine conversion of base 2854 that was present only in af- healthy and fertile. A comprehensive histopathological analysis of fected Lym1 mice (Fig. 1D). This base change is predicted to cause these mice, however, revealed an absence of peripheral LN (in- the substitution of a stop codon for Y868, truncating the protein in guinal, brachial, axillary, cervical) and Peyer’s patches (Fig. 1B the C-terminal phosphorylation site (Fig. 1E). This led to an NF- and data not shown). Mesenteric LN were present, but were re- ␬B2 precursor protein with a predicted mass of 96 kDa, termed duced in size and cellularity (data not shown). p100Lym1 (Fig. 1F). The Lym1 mutation introduced a Bsu 36I re- striction enzyme recognition site, enabling Lym1 mice to be geno- Lym1 is an allele of Nfkb2 and encodes a truncated protein typed using RFLP analysis (Fig. 1G). The Lym1 mutation was genetically mapped by mating affected Lym1 mice on a BALB/c background with C57BL/6 wt mice and then intercrossing the F progeny. DNA from both affected and Disrupted secondary lymphoid organ development and 1 inflammation in Nfkb2Lym1 mice unaffected F2 mice was typed for a genome-wide panel of poly- morphic simple sequence length polymorphism markers. The mu- Upon dissection, it was found that Nfkb2Lym1/Lym1 mice lacked tation was localized to a 6-Mb interval on chromosome 19 (Fig. mesenteric LNs in addition to peripheral LNs and Peyer’s patches 1C). The Nfkb2 gene, located within this region, was considered a (data not shown), suggesting that the Lym1 mutation led to a more strong candidate as LN development is perturbed in Nfkb2Ϫ/Ϫ severe phenotype in the homozygous state. Nfkb2Lym1/Lym1 mice mice (9, 10). also displayed reduced fertility compared with either wt or The Journal of Immunology 7519

Nfkb2Lym1/ϩ mice, with less frequent litters and smaller litter sizes (data not shown). Disorganized splenic architecture is a hallmark of mouse models in which the noncanonical NF-␬B pathway is disrupted, including Nfkb2Ϫ/Ϫ mice (9, 10, 22). In spleen sections from Nfkb2Lym1/Lym1 mice, T and B cells were not segregated into discrete compart- ments and the number of follicular dendritic cell clusters and mar- ginal zone B cells were severely reduced (Fig. 2, A–C). In spleen from wt mice, marginal metallophillic macrophages, defined by staining with MOMA-1, formed a ring-like zone defining the mar- ††† ginal zone, but this structure was not evident in Nfkb2Lym1/Lym1 spleen (Fig. 2D). The abnormalities in splenic architecture in Nfkb2Lym1/ϩ mice were intermediate between that of wt and Nfkb2Lym1/Lym1 mice (data not shown). Thus, although peripheral blood lymphocytosis is inherited as a dominant trait, disruptions to LN and spleen development are inherited as semidominant phenotypes. Histological analysis showed the presence of inflammatory cell in- µ

filtrates in the lung and liver of mice carrying the Lym1 mutation (Fig. Downloaded from 2 and data not shown). In Nfkb2Lym1/Lym1 mice, these infiltrations were extensive with large foci composed of T and B cells, and mac- rophages (Fig. 2, E–H). Smaller foci of similar composition were present in the lung and liver of Nfkb2Lym1/ϩ mice (data not shown). Inflammatory foci are seen in other mouse models with per- FIGURE 5. Osteopetrosis in Nfkb2Lym1 mice. A, In vitro osteoclasto-

turbed noncanonical pathway signaling (11, 34–36), which may genesis assays showing the number of tartrate-resistant acid phospha- http://www.jimmunol.org/ reflect inadequate self-tolerance due to reduced Aire expression tase (TRAP)-positive multinucleated cells (MNC) generated from and disorganized medullary thymic epithelial cell networks (13, RANKL-stimulated primary bone marrow cultures. Histomorphometry showing trabecular bone volume (B) number (C), and thickness (D). Ͻ ءء Indeed, Aire expression was significantly reduced in the Ϯ .(36 ,14 Lym1/Lym1 Data are mean SEM of three to five mice of each genotype. , p p Ͻ 0.001 relative to wt mice; †, p Ͻ 0.001 Nfkb2Lym1/ϩ ,ءءء ;thymus of Nfkb2 mice, suggesting that the inflammatory 0.01 lesions evident in the lung and liver of these mice are due to relative to Nfkb2Lym1/Lym1 mice. autoimmune processes (Fig. 2I). There was a progressive in- crease in severity of the inflammation as Nfkb2Lym1/Lym1 mice aged (data not shown), which likely contributed to their reduced not shown), suggesting that splenomegaly resulted from an expan- lifespan (Fig. 2J). sion of the splenic red pulp. by guest on September 23, 2021 Perturbed T cell development in Nfkb2Lym1/Lym1 mice Impaired B cell maturation and function in Nfkb2Lym1 mice T cell development in the thymus of Nfkb2Lym1 mice was analyzed We next analyzed B cell development in the bone marrow and by flow cytometry. Overall, the thymus was enlarged in young spleen. The proportion and number of pro-B, pre-B, and immature Nfkb2Lym1/Lym1 mice compared with wt, but became smaller in size B cells in the bone marrow was unchanged, suggesting that early and cellularity compared with controls as the mice age and became B cell development is normal in Nfkb2Lym1/Lym1mice (Fig. 4A). moribund (Fig. 3A and data not shown). The total number of CD8 The number of mature recirculating B cells was significantly re- single positive (SP) cells was unchanged in Nfkb2Lym1/Lym1 thymus duced, however, in Nfkb2Lym1/Lym1 bone marrow compared with compared with wt controls (Fig. 3A). Double positive, double neg- wt (Fig. 4A). The number of transitional stage 1 B cells was ative, and CD4 SP cells, however, were significantly increased in significantly increased and there was a profound reduction in number (Fig. 3A). An increased proportion of the CD4 SP cells had the number of transitional stage 2 B cells, marginal zone, and the appearance of recent thymic emigrants (CD69Ϫ, CD62Lϩ) follicular B cells in Nfkb2Lym1/Lym1 spleens (Fig. 4B). Measure- (13, 37), suggesting that thymic export may be impaired in ment of serum Ig in resting Nfkb2Lym1/Lym1 mice showed a signif- Nfkb2Lym1/Lym1 mice (Fig. 3A, bottom). icant reduction in the basal levels of IgM, IgG1, IgG2b, and IgA The blood of Nfkb2Lym1/Lym1 mice contained a significantly in- compared with wt (Fig. 4C). Proliferation of purified B cells from creased number of CD4 and CD8 T cells compared with wt, and a Nfkb2Lym1/Lym1 mice in response to CD40L, LPS, or BCR ligation small increase in B cell number, although this increase was not was significantly reduced (Fig. 4D). Induction of isotype switching significant (Fig. 3B). There was no major difference in the propor- and plasma cell differentiation in vitro in response to these same tion of CD69Ϫ CD62Lϩ peripheral blood or splenic CD4 cells stimuli was also reduced in Nfkb2Lym1/Lym1 B cells (data not between wt and Nfkb2Lym1/Lym1 mice (Fig. 3C and data not shown). shown). These defects are more likely a consequence of decreased There was no difference in the expression of CD25 between wt and B cell proliferation or survival (Fig. 4E) rather than an intrinsic Nfkb2Lym1/Lym1 T cells in all organs tested, but the expression of inability to differentiate, as Nfkb2Lym1/Lym1 B cells were capable of CD44 was slightly reduced, suggesting that the T cells were more differentiation in vitro, but at reduced frequency (data not shown). naive in Nfkb2Lym1/Lym1 mice (data not shown). Similar defects in B cell development and function are character- Ϫ Ϫ T cells were represented in normal number in the spleen, but istic of Nfkb2 / and aly mice (9, 10, 15, 38). Lym1/Lym1 spleens were enlarged in Nfkb2 mice (Fig. 3C). This Lym1 splenomegaly did not correlate with a significant increase in total Osteoclastogenesis is impaired in Nfkb2 mice splenocyte number (Fig. 3C) and became more pronounced as the The noncanonical NF-␬B pathway is known to be critical for the mice age (data not shown). Histological staining showed an in- differentiation of osteoclasts via RANKL stimulation (24). To assess crease in erythropoiesis in the spleen of Nfkb2Lym1/Lym1 mice (data the effect of the Lym1 mutation on this process, we cultured the 7520 A NONPROCESSIBLE MUTANT FORM OF NF-␬B2

FIGURE 6. Inhibition of NF-␬B signaling pathways in Nfkb2Lym1 cells. A, Cytoplasmic fractions of B cells untreated (Ϫ) or stimulated (ϩ) with anti-CD40 for 4 h were blotted with an NF-␬B2 Ab. B, Nuclear extracts of purified B cells stimulated as in A were blotted with Abs specific for NF- ␬ B2 and RelA. C, Mouse embryonic Downloaded from fibroblasts were untreated (Ϫ) or stim- ulated (ϩ) with anti-LT␤RAbfor 15 h, immunoprecipitated with RelA- specific Ab, and blotted with anti- p100 and anti-RelA. D, Cytoplasmic fractions of bone marrow macro-

phages untreated (Ϫ) or stimulated β http://www.jimmunol.org/ with LPS (ϩ) were blotted with Abs specific for either RelA or NF-␬B1 (p105/p50). Band intensity was quan- titated and normalized to each loading control (bottom panels). Data are rep- resentative of at least two independent experiments. by guest on September 23, 2021

β

bone marrow of mutant mice in RANKL and M-CSF to stimulate p100Lym1, both S870 and S872 are predicted to be absent, sug- osteoclast formation. The ability of Nfkb2Lym1/ϩ cells to form os- gesting that p100Lym1 is unable to be phosphorylated at the C teoclasts in response to RANKL stimulation was significantly re- terminus. Phosphorylation of p100 leads to recruitment of the E3 duced, and Nfkb2Lym1/Lym1 bone marrow was almost completely ubiquitin ligase ␤ transducin repeat-containing protein ␤TrCP unresponsive to RANKL with osteoclast formation Ͻ0.5% of that (40), which transfers ubiquitin to K855, thus targeting the precur- seen with wt (Fig. 5A). Consistent with this response, histomor- sor p100 form of NF-␬B2 for processing to p52 by the proteasome. phometric analysis of Nfkb2Lym1/Lym1 bones showed a mild osteo- Thus, it appeared likely that processing of the NF-␬B2 precursor petrosis, with significantly increased trabecular bone volume (Fig. protein would be perturbed in mice carrying the Lym1 mutation. 5B) and number (Fig. 5C) but no change in trabecular thickness To test this likelihood, we stimulated purified splenic B cells (Fig. 5D), indicating a corresponding defect in basal osteoclasto- with anti-CD40 Ab. Cytoplasmic and nuclear fractions were pre- genesis in vivo. There was, however, no obvious defect in tooth pared and Western blotted to analyze the activation and nuclear eruption in Nfkb2Lym1/Lym1 mice (data not shown). translocation of NF-␬B proteins. Both p100 and p52 were detect- able in cytoplasmic extracts from untreated and stimulated wt B ␬ The Lym1 mutation prevents processing of the NF- B2 cells (Fig. 6A). Cytoplasmic p100 levels were reduced after stim- precursor protein ulation, suggesting that p100 was processed into p52, which mi- The premature stop codon introduced by the Lym1 mutation trun- grated to the nucleus (Fig. 6A, bottom). In Nfkb2Lym1/ϩ cells, little, cates p100Lym1 at Y868 in the C-terminal phosphorylation se- if any, p52 was detectable in lysates from either untreated or stim- quence (Fig. 1D). Previous studies have shown that S866 and S870 ulated cells and there was no apparent reduction in precursor levels comprise the docking site for the upstream kinase, IKK␣ (39), and upon stimulation. In Nfkb2Lym1/Lym1 cells, however, p100Lym1 ac- S872 is phosphorylated by IKK␣ (39). Although S866 is present in cumulated in response to anti-CD40 stimulation, and no p52 was The Journal of Immunology 7521

observed in either the presence or absence of stimulation, even ported recently (26). Lymphocytosis did not appear to contrib- with longer film exposures (Fig. 6A and data not shown). ute to the development of splenomegaly in Nfkb2Lym1/Lym1 Immunoblotting of nuclear extracts from wt B cells showed both mice, however, and similar to RelbϪ/Ϫ mice, this appears to be p52 and RelA were recruited to the nucleus following stimulation due to increased erythropoiesis (34). (Fig. 6B). In contrast, only minor increases in nuclear p52 or RelA Similar to Nfkb2Ϫ/Ϫ and aly mice, Nfkb2Lym1/Lym1 mice have ϩ were seen in Nfkb2Lym1/ cells following stimulation (Fig. 6B). In disrupted splenic architecture and B cell differentiation (9, 10). resting mouse embryonic fibroblasts, p100 associated with RelA in Unlike the defect in B cell differentiation in Nfkb2Ϫ/Ϫ mice, which both wt and Nfkb2Lym1/Lym1 cells (Fig. 6C). After stimulation of wt is first apparent after the transitional stage 1 B cell maturation in cells with an agonist Ab specific for LT␤R, this association was the spleen (15), there is a significant increase in the number of substantially reduced, most likely as p100 was processed to p52. In splenic transitional stage 1 B cells in Nfkb2Lym1/Lym1 mice, sug- Lym1 contrast, the amount of p100 associated with RelA in gesting that B cell differentiation may be blocked at this stage. The Lym1/Lym1 Nfkb2 cells increased upon stimulation (Fig. 6C). Simi- extent of B cell deficiency of more mature subsets, however, is larly, nuclear transport of RelA and p50 in response to LPS stim- similar between Nfkb2Ϫ/Ϫ and Nfkb2Lym1/Lym1 mice (15). The ap- ulation through the canonical NF-␬B pathway appeared to be in- pearance of these phenotypes in Nfkb2Ϫ/Ϫ mice suggests that p52 Lym1/Lym1 hibited in Nfkb2 cells, as cytoplasmic RelA levels failed plays a critical and nonredundant role in the maintenance of to decrease and p50 levels accumulated in response to LPS stim- splenic architecture and B cell differentiation, and that other ulation, in contrast to wt cells (Fig. 6D). NF-␬B proteins cannot compensate for the loss of p52. In contrast, Lym1/Lym1 Thus, in resting wt and Nfkb2 cells, p100 appears to osteoclastogenesis and LN formation are only mildly perturbed in bind RelA and inhibit its activation and nuclear translocation. In wt Nfkb2Ϫ/Ϫ mice (16, 17, 24), suggesting that p50:RelA activation Downloaded from cells, this inhibition is relieved upon stimulation through the non- may contribute to these processes. In the presence of the nonproc- ␬ canonical NF- B pathway as p100 is processed to p52. The in- essible NFkB2 allele in Nfkb2Lym1/Lym1 mice, RelA activation is Lym1 Lym1/Lym1 ability to process p100 in Nfkb2 cells, however, re- limited, reducing the extent of NF-␬B signaling to below the sults in a “super repressor” form of p100 that not only prevents the threshold necessary for LN formation and osteoclastogenesis and formation of p52, but also continually inhibits RelA activation and hence abnormalities result. Alternatively, retention of RelB in the nuclear translocation, regardless of stimulation with ligands that cytoplasm by p100Lym1 may contribute to abnormalities in LN http://www.jimmunol.org/ activate either the canonical or noncanonical NF-␬B pathway. Ϫ Ϫ development because Relb / mice lack all LN (34). The phenotype of Nfkb2Lym1/Lym1 mice resembles that of mice in Discussion which loss of NIK function prevents NF-␬B2 processing (aly or NF-␬B2 is a critical mediator of the noncanonical NF-␬B pathway. Ϫ Ϫ Ϫ Ϫ Nik / mice) (21, 22). The similarities between Nik / and The importance of NF-␬B2 in the development and function of a Nfkb2Lym1/Lym1 mice suggest that NIK function is largely limited to variety of organs and cell lineages has been highlighted by the the induction of p100 processing. Some differences, however, are study of genetically modified mice in which NF-␬B2 function has seen between these mouse models. Although RANKL-stimulated been ablated or modified (9, 10, 18, 21, 22, 24). In this study, we

osteoclastogenesis in vitro is impaired in both models, basal oste- by guest on September 23, 2021 describe the phenotype of mice harboring a novel mutation in oclastogenesis in vivo is normal in NikϪ/Ϫ mice, but is signifi- Nfkb2, which encodes a nonprocessible form of p100. We have cantly perturbed in Nfkb2Lym1/Lym1 mice (24). The cause of this shown that the abnormalities in Nfkb2Lym1/Lym1 mice are more se- ␬ discrepancy is unclear. vere than those seen in mice completely lacking NF- B2, indicat- Lym1/Lym1 Ϫ/Ϫ ␬ Nfkb2 mice also present some similarities with Nfkb1 / ing a role for NF- B2 in the regulation of both the canonical and Ϫ/Ϫ ␬ Nfkb2 mice. Although partial defects in LN formation are noncanonical NF- B pathways. Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Nfkb2Lym1/Lym1 mice develop inflammatory lesions in the liver seen in Nfkb2 mice, all LNs are absent in Lta , Ltbr , NikϪ/Ϫ, and Nfkb1Ϫ/Ϫ/Nfkb2Ϫ/Ϫ mice (11, 17, 22, 23). Similarly, and lung that are reminiscent of those observed in mice deficient in Ϫ/Ϫ Ϫ/Ϫ RelB, LT␤R, or functional NIK (11, 34, 41). We have shown that Nfkb1 /Nfkb2 mice have severe osteopetrosis, whereas sin- Aire expression is significantly reduced in the thymus of gle knockout mice have no obvious bone defects (42), providing Nfkb2Lym1/Lym1 mice, suggesting that a breakdown in peripheral further evidence that p100 regulates the activation of p50:RelA tolerance is a contributing factor. A recent study has shown that complexes. Indeed, p100 has been shown to interact with p50: RANK signals regulate the development of Aire-expressing med- RelA and regulate RelA activation in a number of previous studies ullary thymic epithelial cells, a process that is likely to be disrupted (24–26, 43, 44). Consistent with this finding, we have shown that in Nfkb2Lym1/Lym1 mice (14). Similar to other mice with defective p100 associates with RelA in resting cells, preventing its nuclear NF-␬B signaling (13), we noticed that the thymic medulla and translocation and activation. Stimulation through the noncanonical cortex of Nfkb2Lym1/Lym1 mice are poorly defined (data not shown), NF-␬B pathway relieves this inhibition by processing p100 to p52 indicating that thymic structure is disorganized. and thus releasing RelA. Lym1/Lym1 The differentiation of thymocytes is also perturbed, with an The defects in Nfkb2 mice, however, are not as severe Ϫ/Ϫ Ϫ/Ϫ increased number of double negative, double positive, and par- as in Nfkb1 /Nfkb2 mice, which die shortly after weaning Lym1/Lym1 ticularly CD4 SP cells, present in the thymus. Despite having and lack all mature B cells (42, 45). Similarly, Nfkb2 Ϫ Ϫ the appearance of recent thymic emigrants, suggesting a block- mice present a milder phenotype than either Rela / (embryonic Ϫ Ϫ ade in thymic emigration as suggested for LtrbϪ/Ϫ and aly mice lethal) or Relb / (multifocal inflammatory disease within 2 wk of (13), this expansion of CD4 T cells was also observed in pe- birth) mice (34, 46), suggesting there is only a partial block in ripheral blood, and together with an increase in peripheral CD8 RelA and RelB signaling in Nfkb2Lym1/Lym1 mice. The increased Ϫ Ϫ T cells, appears to account for the lymphocytosis evident in the severity of the Nfkb2Lym1/Lym1 phenotype compared with Nfkb2 / blood of Nfkb2Lym1/Lym1 mice. Peripheral blood lymphocytosis mice likely reflects the constitutive inhibitory effect of p100Lym1 has not been widely reported in other mouse models with ab- on canonical pathway signaling. Activation of canonical pathway normal NF-␬B signaling, and it is unclear what is driving this signaling induces p100 expression, leading to accumulation of defect, although it could reflect hyperactivity of Nfkb2Lym1/Lym1 p100Lym1 and retention of both canonical and noncanonical bind- naive CD4 T cells in response to TCR stimulation, as was re- ing partners in the cytoplasm. Given the similarities in phenotype 7522 A NONPROCESSIBLE MUTANT FORM OF NF-␬B2

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