Impairment of Mature B Cell Maintenance upon Combined Deletion of the Alternative NF-κB Transcription Factors RELB and NF- κB2 in B Cells This information is current as of September 29, 2021. Nilushi S. De Silva, Kathryn Silva, Michael M. Anderson, Govind Bhagat and Ulf Klein J Immunol published online 5 February 2016 http://www.jimmunol.org/content/early/2016/02/04/jimmun ol.1501120 Downloaded from

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 5, 2016, doi:10.4049/jimmunol.1501120 The Journal of Immunology

Impairment of Mature B Cell Maintenance upon Combined Deletion of the Alternative NF-kB Transcription Factors RELB and NF-kB2 in B Cells

Nilushi S. De Silva,*,† Kathryn Silva,* Michael M. Anderson,* Govind Bhagat,*,‡ and Ulf Klein*,†,‡

BAFF is critical for the survival and maturation of mature B cells. BAFF, via BAFFR, activates multiple signaling pathways in B cells, including the alternative NF-kB pathway. The transcription factors RELB and NF-kB2 (p100/p52) are the downstream mediators of the alternative pathway; however, the B cell–intrinsic functions of these NF-kB subunits have not been studied in vivo using conditional alleles, either individually or in combination. We in this study report that B cell–specific deletion of relb led to

only a slight decrease in the fraction of mature splenic B cells, whereas deletion of nfkb2 caused a marked reduction. This Downloaded from phenotype was further exacerbated upon combined deletion of relb and nfkb2 and most dramatically affected the maintenance of marginal zone B cells. BAFF stimulation, in contrast to CD40 activation, was unable to rescue relb/nfkb2-deleted B cells in vitro. RNA-sequencing analysis of BAFF-stimulated nfkb2-deleted versus normal B cells suggests that the alternative NF-kB pathway, in addition to its critical role in BAFF-mediated cell survival, may control the expression of genes involved in the positioning of B cells within the lymphoid microenvironment and in the establishment of T cell–B cell interactions. Thus, by ablating the

downstream transcription factors of the alternative NF-kB pathway specifically in B cells, we identify in this study a critical http://www.jimmunol.org/ role for the combined activity of the RELB and NF-kB2 subunits in B cell homeostasis that cannot be compensated for by the canonical NF-kB pathway under physiological conditions. The Journal of Immunology, 2016, 196: 000–000.

he requirement of tonic signaling through the BCR for the ERK, protein kinase Cb, and PI3K signaling axes that sustain maintenance of resting, mature B cells is well established B cell survival and metabolic fitness (12–15). In addition, BAFF- T (1, 2). A second crucial survival signal for mature B cells mediated activation of the NF-kB signaling pathway is a major is mediated via the binding of BAFF to BAFFR, which is regulator of B cell maintenance (16, 17). expressed on B cells (3). Genetic ablation of either BAFF or The NF-kB signaling pathway can be divided into two major BAFFR results in a dramatic reduction of peripheral B cells (4–9), branches, a canonical and an alternative pathway that are activated by guest on September 29, 2021 whereas BAFF overexpression causes B cell hyperplasia (10, 11). through specific cell surface receptors (18, 19). The downstream BAFF or BAFFR deficiencies lead to B cell maturation arrest at mediators of the canonical pathway are the transcription factors the transitional 1 (T1) developmental stage (3). As a result, the c-REL, RELA, and p50 that mainly occur as heterodimers. In B cells, survival of follicular B cells is strongly impaired and marginal these heterodimers translocate from the cytoplasm to the nucleus zone (MZ) B cell development is ablated. BAFF activates several in response to BCR, TLR, and CD40 stimulation (20, 21). The cor- downstream signaling routes in B cells. It stimulates the AKT, responding downstream mediators of the alternative pathway are RELB (encoded by relb) and p52 (generated by proteolytic cleav- age of its precursor p100, encoded by nfkb2). These subunits mainly *Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY † occur as heterodimers and in B cells are activated by soluble BAFF 10032; Department of Microbiology and Immunology, Columbia University, New York, NY 10032; and ‡Department of Pathology and Cell Biology, Columbia Uni- or CD40 stimulation via interaction with T cells expressing CD40L versity, New York, NY 10032 (20–22). RELB, but not p52, contains a transactivation domain and ORCID: 0000-0001-6250-048X (G.B.). is thus capable of activating transcription. Receptor-mediated acti- Received for publication May 15, 2015. Accepted for publication January 5, 2016. vation of the alternative pathway leads to the release of NF-kB– This work was supported by National Cancer Institute/National Institutes of Health inducing kinase (NIK) from the TRAF2/TRAF3/c-IAP1/2 complex, Grant R01-CA157660 (to U.K.), a grant from the Stewart Trust Foundation, the allowing its stabilization (23). NIK activates IkB kinase-a (IKKa), Herbert Irving Comprehensive Cancer Center, and a Cancer Biology Training Pro- gram fellowship (National Cancer Institute/National Institutes of Health 19 Grant which phosphorylates and subsequently induces cleavage of p100 5T32-CA009503-26 to N.S.D.S.). that is bound to RELB. This results in both the generation of p52 The RNA-sequencing data have been submitted to the Gene Expression Omni- and the release of RELB, thereby allowing the nuclear translocation bus (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE75761 and of RELB/p52 heterodimers (24). GSE75762. In resting, mature B cells, the predominant route of BAFF- Address correspondence and reprint requests to Dr. Ulf Klein, Herbert Irving Com- mediated NF-kB activation is via the alternative pathway (25– prehensive Cancer Center, Columbia University, 1130 St. Nicholas Avenue, R312, New York, NY 10032. E-mail address: [email protected] 27). There is, however, evidence for an additional contribution of The online version of this article contains supplemental material. the canonical pathway to BAFF-mediated prosurvival functions k Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; eGFP, enhanced (28, 29), and constitutive canonical NF- B signaling can fully GFP; ES, embryonic stem; IKKa,IkB kinase-a; MZ, marginal zone; NIK, NF-kB– replace BAFFR signals (30). A role for the alternative pathway in inducing kinase; PI, propidium iodide; RNA-seq, RNA-sequencing; T1, transitional B cell homeostasis has previously been demonstrated based on the 1; T2, transitional 2; T3, transitional 3; WT, wild-type. analysis of mice deficient in upstream components of the pathway Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 (31–38). Whereas these studies have identified NIK and IKKa as

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501120 2 CELL-INTRINSIC ROLES OF RELB AND NF-kB2 IN B CELLS critical factors in inducing the processing of p100 that facilitates 2.0 kb of the region upstream of the nfkb2 promoter region; the nfkb2 nuclear translocation of RELB/p52 heterodimers, they can have promoter region, exon 1 and exon 2, which contains the translational start additional functions. In certain cell systems, NIK has been found site (overall 2.4 kb); and 4.6 kb of the region downstream of exon 2. The a linearized vectors were electroporated into KV1 embryonic stem (ES) to also activate the canonical pathway (39–41), and IKK has cells (a 129:B6 hybrid ES cell line), and correctly targeted ES cell colonies known NF-kB–independent roles (42). These alternative NF-kB were identified by Southern blot analysis after selection with gancyclovir pathway–independent functions of NIK and IKKa make it diffi- and G418 (Supplemental Fig. 1). Chimeras were obtained after injection of cult to conclusively identify the biological role of RELB/p52- targeted ES cell clones into blastocysts derived from C57BL/6 mice. From the chimeras bred with C57BL/6 females, we obtained mice with the mediated target gene transcription from these studies. conditional relb and nfkb2 alleles in the germline. The conditional relb and The identification of the lymphocyte-intrinsic functions of RELB nfkb2 alleles were backcrossed to C57BL/6 mice (n $ 7). CD19-Cre mice and NF-kB2 (that is, both p52 and p100) has been hampered by the have been described (49). Mice were housed and treated in compliance fact that constitutional relb and nfkb2 knockout mice show severe with the Department of Health and Human Services Guide for the Care defects in lymphoid organization due to the lack of RELB or and Use of Laboratory Animals and according to the guidelines of the k Institute of Comparative Medicine at Columbia University. The animal NF- B2 in stromal cells (43–45). Moreover, although the individual protocol was approved by the Institutional Animal Care and Use Com- roles of RELB and NF-kB2 in mature B cell development have mittee of Columbia University. been investigated with radiation-induced chimeras generated by transplanting hematopoietic cells from relb2/2 mice or by adoptive B cell isolation and culture 2 2 transfer of bone marrow from nfkb2 / mice into RAG1-deficient Single-cell suspensions of mouse spleen were subjected to hypotonic animals (44, 46), similar experiments with hematopoietic cells from lysis, and B cells were purified by depletion of magnetically labeled non- 2 2 2 2 relb / nfkb2 / mice to determine the biological consequences of B cells using the MACS B cell isolation kit (Miltenyi Biotec). Purified Downloaded from k B cells from the indicated genotypes were cultured in the presence of the combined deletion of RELB and NF- B2 in B cells have not 1 mg/ml anti-mouse CD40 (clone HM40-3; BD Pharmingen) or 25 ng/ml been conducted. BAFF (R&D Systems). Cell density in CD40 and BAFF stimulation ex- The studies outlined above have revealed much about the role periments was 1.5 3 106 cells/ml. For the RNA-sequencing (RNA-seq) of the alternative NF-kB pathway in BAFF-mediated B cell ho- analysis, cell pellets were lysed with TRIzol reagent (Life Technologies) for RNA isolation. For Western blot analysis, purified B cells were washed meostasis; however, several issues remain to be clarified. First, the once with PBS and subjected to Nonidet P40-based lysis. cell-intrinsic requirement of the NF-kB2 or RELB subunits for http://www.jimmunol.org/ mature B cell maintenance has not been determined using con- Flow cytometry ditional alleles that can be deleted specifically in B cells. Second, Spleen cell suspensions or cultured B cells were stained on ice in PBS/0.5% knockout of either relb or nfkb2 alone does not allow complete BSA with the following Abs. From BD Pharmingen: allophycocyanin- ablation of the alternative NF-kB pathway. Indeed, in the absence conjugated anti-IgM (clone II/41), PE-conjugated anti-IgD (clone 11- of either relb or nfkb2, the remaining alternative NF-kB2 subunit 26c.2a), and PE-conjugated anti-CD23 (clone B3B4). From BioLegend: may be able to direct transcription by binding to canonical NF-kB PerCP-conjugated anti-B220 (clone: RA3-6B2), allophycocyanin-conjugated anti-CD21 (clone: 7E9), PE-conjugated anti-b2 (clone: M18/2), PE- subunits. Furthermore, in nfkb2 knockout mice, lack of p100 not conjugated anti-CD93 (clone: AA4.1), biotin-conjugated anti-ICOSL only prevents the generation of p52, but also eliminates the p100 (clone: HK5.3) followed by streptavidin-PerCP (BD Pharmingen), PE- inhibitor. p100 retains RELB in the cytoplasm, and, in its absence, conjugated anti-BAFFR (clone: 7H22-E16), and PercP/Cy5.5-conjugated by guest on September 29, 2021 inappropriate translocation of RELB into the nucleus may occur, anti-CD40 (clone: 3/23). Annexin V/7-aminoactinomycin D (7-AAD) stainings were conducted using the allophycocyanin Annexin V Apoptosis resulting in target gene transcription via the formation of hetero- Detection Kit with 7-AAD (BioLegend). For DNA content analysis, cells dimers with other NF-kB subunits. Also, recent evidence suggests were lysed and stained with propidium iodide (PI). The cells or nuclei were that RELB and p52 have common, but also distinct DNA binding analyzed on a FACSCalibur or LSRII (BD Biosciences). Data were ana- sites in the genome (47); how these sites would be modulated lyzed using FlowJo software. k upon binding of alternative NF- B subunits dimerizing with ca- Immunohistochemistry nonical subunits is unclear. Therefore, identification of the bio- m logical consequences of complete inactivation of the alternative Sections of 3 m in thickness were cut from spleen tissue that was fixed k k overnight in 10% formalin and embedded in paraffin. Sections were NF- B pathway requires ablation of both RELB and NF- B2 stained with either H&E, unlabeled anti-CD3 rabbit Ab (clone: SP7; subunits. To address these issues, we have generated conditional Thermo Scientific), or anti-GFP rabbit Ab (Molecular Probes; Invitrogen). relb and nfkb2 alleles. We found that combined deletion of relb Abs were incubated overnight at 4˚C, stained with anti-rabbit HRP-labeled and nfkb2 in B cells had a markedly stronger effect on the survival polymer (Dako), and developed in aminoethylcarbazole (Sigma-Aldrich). of mature B cells compared with the single gene deletions. To These slides were then counterstained for IgM by overnight incubation at 4˚C with alkaline peroxidase–conjugated anti-IgM Ab (Southern Biotech). our knowledge, these results for the first time reveal the extent IgM stainings were developed in NBT/5-bromo-4-chloro-3-indolyl phos- to which the activity of the alternative NF-kB pathway controls phate (Roche, New York, NY). The images were acquired by means of a B cell homeostasis in vivo, and provide new insights into the bi- Digital Sight camera (Nikon) mounted on a Nikon Eclipse E600 micro- ological program controlled by the alternative NF-kB pathway in scope (Nikon). response to BAFF. Immunoblot analysis Purified B cells were subjected to Nonidet P40-based lysis, separated by Materials and Methods SDS-PAGE, and blotted on nitrocellulose membranes (GE Healthcare). Generation of mice carrying a floxed relb or nfkb2 allele Samples were incubated with the following primary Abs overnight at 4˚C: rabbit anti-RELB (Santa Cruz, clone: C-19), rabbit anti-p100/p52 (Cell The vector to target relb and nfkb2 has been described previously in the Signaling Technologies; CST), mouse anti–b-actin (Sigma-Aldrich; clone: context of the targeting of the irf4 gene (48). The vector was constructed AC-15), rabbit anti-pAKT (Cell Signaling; clone: D9E), and rabbit anti- such that, upon Cre-mediated deletion, the promoter regions and the exons AKT (CST). HRP-conjugated secondary Abs and ECL Western blotting comprising the first ATG of relb or nfkb2 were deleted with simultaneous Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo activation of enhanced GFP (eGFP) expression (Supplemental Fig. 1). Scientific) were used for detection. Successively inserted into the cloning sites of the vector were each three DNA fragments of the relb and nfkb2 loci comprising the following: 1) RNA-seq relb, 2.2 kb of the region upstream of the relb promoter region; the relb promoter region, exon 1, which contains the translation start site and exon RNA was harvested from three culture replicates/genotype of BAFF- 2 (overall 2.8kb); and 3.8 kb of the region downstream of exon 2. 2) nfkb2, stimulated B cells and three culture replicates/genotype of CD40- The Journal of Immunology 3 stimulated B cells and submitted for RNA sequencing at the Columbia nfkb2 ES cell lines were identified by a PCR-based screening University Genome Center. Thirty million single-end, 100-bp reads were strategy, followed by confirmation via Southern blot analysis sequenced per sample on the Illumina HiSeq2000/2500 V3 instrument. (Supplemental Fig. 1A, 1B). DESeq analysis was used to identify differentially expressed genes and fl/fl determine fold expression changes between genotypes. RNA-seq data are Western blot analysis of B cells isolated from relb CD19-Cre available through the Gene Expression Omnibus database (http://www.ncbi. and nfkb2fl/flCD19-Cre mice demonstrated that the protein levels nlm.nih.gov/geo/) under accession numbers GSE75761 and GSE75762. of RELB and p52, respectively, were strongly reduced compared Statistical analysis with B cells from littermate controls (Fig. 1B, 1E), confirming the loss of functionality of the newly generated loxP-flanked relb and The p values were obtained using unpaired Student t test or by one-way nfkb2 alleles. The remaining protein is likely to be derived from ANOVAwith Tukey’s multiple comparisons (GraphPad Prism 5 software). 2 The p values ,0.05 were considered significant. eGFP B cells that have escaped Cre-mediated deletion. This possibility was confirmed by the complete absence of RELB + fl/fl Results protein in flow-sorted eGFP B cells from relb CD19-Cre mice (Fig. 1C) (the low eGFP expression in B cells of nfkb2fl/flCD19- Conditional deletion of relb and nfkb2 in B cells Cre mice prevented us from unambiguously separating eGFP+ and To determine the B cell–intrinsic functions of RELB and NF-kB2 eGFP2 B cells; we were therefore unable to perform a similar in the maintenance of B cells in vivo, we generated transgenic experiment for NF-kB2). B cells from relbfl/+CD19-Cre and mouse strains carrying loxP-flanked alleles of relb and nfkb2 nfkb2fl/+CD19-Cre showed reduced protein expression of RELB (encoding p100/p52), respectively (Fig. 1A, 1D). To enable and p52, respectively, relative to the CD19-Cre controls, indicat-

B cell–specific ablation of relb and nfkb2, these mice were crossed ing haploinsufficiency of RELB and p52 expression in B cells Downloaded from to mice expressing Cre recombinase specifically in B cells (49). (Fig. 1B, 1C, 1E). Importantly, in the absence of Cre, the The region of the relb or nfkb2 genes flanked by loxP sites unrearranged conditional relb and nfkb2 alleles produced physi- comprised the promoter region and the exon encoding the trans- ological amounts of RELB and p52 protein, respectively (Fig. 1B, lational start site, thus allowing the creation of relb or nfkb2 null 1E). alleles upon Cre-mediated recombination of loxP sites. In addi- Reduced fractions of splenic B cells in the absence of

tion, the conditional alleles were constructed such that the re- http://www.jimmunol.org/ k combination of loxP sites is accompanied by the expression of an alternative NF- B subunits eGFP to enable tracking of relb or nfkb2-deleted cells in the tis- To assess the B cell–intrinsic functions of alternative NF-kB sues. To achieve this, an eGFP minigene was placed upstream of subunits in B cell maintenance, we determined the fraction of the relb or nfkb2 promoter regions in the opposite orientation; this mature splenic B cells present in mice with B cell–specific abla- minigene is silent in the targeted locus. However, following re- tion of RELB or NF-kB2 individually, or combined RELB and combination of loxP sites, a phosphoglycerate kinase promoter NF-kB2 inactivation. Upon B cell–specific ablation of RELB (placed in intron 2 of relb or nfkb2) is placed proximal to the in relbfl/flCD19-Cre mice, we observed a slight but significantly eGFP gene, allowing transcription. A similar strategy was previ- reduced fraction of splenic B cells compared with relbfl/+CD19- ously used for the conditional deletion of irf4, rel, and rela (48, Cre and CD19-Cre control mice (Fig. 2A; for cell numbers, see by guest on September 29, 2021 50). In this system, gene deletion is directly linked to induction of Supplemental Fig. 2). B cell–specific ablation of NF-kB2 was eGFP expression, and therefore eGFP positivity is a marker for associated with a marked reduction in the fraction of splenic gene deletion. In addition, eGFP expression is quantitative, as B cells; B220+ cells comprised 28% of total splenocytes in nfkb2fl/fl B cells from mice heterozygous or homozygous for the floxed CD19-Cre mice compared with 46 and 50% in nfkb2fl/+CD19-Cre allele can be distinguished by their mean eGFP fluorescence (48, and CD19-Cre control mice, respectively (Fig. 2B; for cell num- 50) (also see Fig. 2A, 2B, lower panel). Correctly targeted relb or bers, see Supplemental Fig. 2). To obtain complete ablation of

FIGURE 1. Generation of mice with conditional deletion of relb or nfkb2 in B cells and simultaneous expression of eGFP. (A and D) Targeting strategy showing the status of relb and nfkb2 before (top) and after (bottom) Cre-mediated recombination. Numbers indicate the respective exons. (B and E) Western blot analysis of RELB and p52 protein levels in purified splenic B cells of the indicated genotypes, and (C) of flow-sorted eGFP+ B cells from relbfl/flCD19- Cre mice and the corresponding eGFP+ relfl/+CD19-Cre and eGFP2 CD19-Cre control mice. 4 CELL-INTRINSIC ROLES OF RELB AND NF-kB2 IN B CELLS

FIGURE 2. Reduced fractions of splenic B cells in the absence of al- ternative NF-kB subunits. (A–C, top) The fractions of splenic B cells from relbfl/flCD19-Cre, nfkb2fl/flCD19-Cre, or relbfl/flnfkb2fl/flCD19-Cre mice and the corresponding heterozygous and CD19-Cre control mice were deter- mined by flow cytometry (for absolute B cell numbers, see Supplemental Fig. 2). Each symbol represents a mouse. Data are shown as mean 6 SD. Sta- tistical significance was determined by one-way ANOVA (***p , 0.001). (A– Downloaded from C, bottom) Flow cytometry of eGFP expression in splenic B cells of the in- dicated genotypes. The number above the gate indicates the percentage of eGFP+ B cells among B220+ Bcellsof relbfl/flnfkb2fl/flCD19-Cre mice. http://www.jimmunol.org/

RELB/p52, the major heterodimer of the alternative pathway, the deficient B cells in these mice, unlike what is observed in relbfl/fl conditional relb and nfkb2 alleles were interbred to generate nfkb2fl/flCD19-Cre mice. Nonetheless, deletion of nfkb2 alone led relbfl/flnfkb2fl/flCD19-Cremicetoallowsimultaneous deletion of to a strong reduction in the fraction of B cells that was consid- by guest on September 29, 2021 relb and nfkb2 in B cells. The fraction of splenic B cells in these erably higher than that observed in relbfl/flCD19-Cre mice, indi- mice was similar to that observed following ablation of NF-kB2 cating that, functionally, p52 appears to be the major subunit alone (B220+ cells comprised 30% of total splenocytes in relbfl/fl downstream of the alternative pathway in B cells. nfkb2fl/flCD19-Cre mice compared with 45 and 50% in relbfl/+ Immunohistochemical staining analysis of spleen sections for nfkb2fl/+CD19-Cre and CD19-Cre control mice, respectively; Fig. IgM and CD3 revealed that, in accordance with the reduced B cell 2C; for cell numbers, see Supplemental Fig. 2). However, analysis fraction observed by flow cytometry, relbfl/flnfkb2fl/flCD19-Cre for eGFP expression among B cells of relbfl/flnfkb2fl/flCD19-Cre mice displayed fewer B cell follicles within the white pulp com- mice identified two distinct eGFP+ and eGFP2 peaks of approx- pared with relbfl/+nfkb2fl/+CD19-Cre and CD19-Cre control mice imately equal proportions (Fig. 2C, bottom panel). The presence (Fig. 3). The size of these B cell follicles was also more hetero- of a sizable eGFP2 population in mice with combined ablation of geneous in relbfl/flnfkb2fl/flCD19-Cre mice compared with the RELB and NF-kB2 indicates that eGFP+ B cells deficient for controls. In summary, combined B cell–specific deletion of relb both RELB and NF-kB2 are outcompeted by eGFP2 B cells that and nfkb2 results in a greatly reduced fraction of B cells that is escaped Cre-mediated deletion. As a result, relb/nfkb2-deleted reflected by abnormalities in splenic white pulp architecture. eGFP+ B cells were present at a frequency of only 15% of total Moreover, the severity of the B cell phenotype upon relb/nfkb2 splenocytes in relbfl/flnfkb2fl/flCD19-Cre mice compared with the deletion appears to surpass that observed with the corresponding B cell fraction of 50% observed in CD19-Cre control mice. single gene deletions. The unique eGFP-expression pattern in relbfl/flnfkb2fl/flCD19- Cre mice contrasts with the single eGFP+ peak observed in Relb/nfkb2-deleted B cells show a developmental block at the B cells from relbfl/flCD19-Cre mice (Fig. 2A, bottom panel), T1 stage suggesting that there is virtually no counterselection against To determine whether the alternative NF-kB subunits are involved RELB-deficient B cells in these mice. Due to the generally low in transmitting BAFF signals during the B cell transitional phase eGFP expression in B cells from nfkb2fl/flCD19-Cre mice, a clear (T1–transitional 3[T3]), where BAFF signaling is known to be distinction between eGFP+ and eGFP2 populations is not possi- required (3), we analyzed splenic B cells from relbfl/flnfkb2fl/fl ble; however, eGFP expression of B cells from nfkb2fl/flCD19- CD19-Cre and control mice for their expression of AA4.1+IgMhigh Cre mice is generally higher than that of B cells from mice CD232 (T1), AA4.1+IgMhighCD23+ (transitional 2 [T2]), and with heterozygous deletion of nfkb2 (nfkb2fl/+CD19-Cre mice) AA4.1+IgMlowCD23+ (T3) B cells. We observed an accumulation (Fig. 2B, bottom panel). In addition, Western blot analysis of T1 B cells and a concomitant reduction in T3 B cells in relbfl/fl (Fig. 1E) demonstrated that the majority of B cells from nfkb2fl/fl nfkb2fl/flCD19-Cre mice in comparison with the CD19-Cre litter- CD19-Cre mice do not express NF-kB2 protein. Together, these mate controls (∼25% versus ∼14% T1 B cells and ∼9% versus observations argue against extensive counterselection of NF-kB2– 18% T3 B cells) (Fig. 4). This finding is in agreement with pub- The Journal of Immunology 5

FIGURE 3. relbfl/flnfkb2fl/flCD19- Cre mice display fewer B cell follicles within the white pulp, and exhibit more heterogeneity in the size of B cell follicles, compared with control mice. Spleen sections from mice of the in- dicated genotypes were analyzed via immunohistochemistry for the expres- Downloaded from sion of CD3 and IgM. One represen- tative mouse of three per group is shown. Original magnification 340 (left) and 3100 (right). http://www.jimmunol.org/ by guest on September 29, 2021 lished data demonstrating that BAFF signaling is required during histochemical staining analysis of splenic sections revealed that the T1 to T2 transition (3) and suggests that the alternative NF-kB the majority of the B cells in the enlarged MZs of relbfl/flnfkb2fl/fl pathway has a role in this transition. CD19-Cre mice did not stain for eGFP, whereas eGFP+ follicular B cells were readily observed (Fig. 5C), a finding that is in ac- Marked counterselection against relb/nfkb2-deleted MZ B cells cordance with the flow cytometry results (Fig. 5B). In summary, fl/fl fl/fl Among the splenic B cell subsets in relb nfkb2 CD19-Cre relbfl/flnfkb2fl/flCD19-Cre mice show strong counterselection against + mice, a slightly reduced fraction of follicular B cells (CD23 relb/nfkb2-deleted MZ B cells. CD21int) and an increased fraction of MZ B cells (CD21high CD232) were observed compared with CD19-Cre mice (∼70% Identification of BAFF-responsive genes controlled by the k versus ∼83% follicular B cells and ∼11% versus ∼6% MZ B cells) alternative NF- B pathway (Fig. 5A). Similar observations were made when analyzing IgMhigh The data outlined above demonstrate that the alternative NF-kB IgDlow and IgM+IgD+ B cell subpopulations as well as CD1d+ and pathway controls a biological program that promotes B cell CD9+ B cell subsets (51, 52) (data not shown). However, the maintenance. To gain insight into this program, we sought to extent of counterselection against relb/nfkb2-deleted B cells dif- identify genes that are transcriptionally regulated by the alterna- fered considerably between the follicular and MZ B cell fractions tive NF-kB subunits in B cells stimulated with BAFF in vitro. A (Fig. 5B), with the strongest counterselection observed in the time-course analysis of BAFF stimulation on normal B cells MZ B cell compartment. Analysis of eGFP expression within MZ revealed that at 6 h, the majority of p100 was processed to p52, B cells (Fig. 5B) demonstrated that only ∼25% of MZ B cells demonstrating robust activation of the alternative NF-kB pathway were eGFP+ compared with ∼57% of follicular B cells. The ob- (Supplemental Fig. 3A). Phosphorylation of AKT, an additional served dramatic counterselection against relb/nfkb2-deleted MZ event known to occur downstream of BAFF stimulation (12), was B cells is consistent with previous publications that invoked roles observed after 24 h of stimulation (Supplemental Fig. 3A). We for RELB or NF-kB2 in the development of MZ B cells from the chose to stimulate B cells for 6 h to identify early BAFF- study of constitutional knockout mice (29, 46). Curiously, mor- responsive genes. B cells purified by magnetic cell separation phologic evaluation of H&E-stained splenic sections from relbfl/fl from nfkb2fl/flCD19-Cre mice were used for this transcriptional nfkb2fl/flCD19-Cre mice demonstrated focal MZ enlargement in profiling experiment, as these mice showed a marked reduction in these mice compared with the controls (data not shown). The mature B cells without evidence of strong counterselection against prominent MZs presumably reflect the higher fractions of MZ nfkb2-deleted B cells, unlike what is observed in relbfl/flnfkb2fl/fl versus follicular B cells that were observed among splenic B cells CD19-Cre mice. B cells isolated from three nfkb2fl/flCD19-Cre by flow cytometric analysis (see Fig. 5A). Importantly, immuno- and CD19-Cre mice each were stimulated with BAFF for 6 h 6 CELL-INTRINSIC ROLES OF RELB AND NF-kB2 IN B CELLS

FIGURE 4. Relb/nfkb2-deleted B cells show a developmental block at the T1 stage. IgM and CD23 expres- sion of AA4.1+ splenic B cells from mice of the indicated genotypes were analyzed by flow cytometry. Numbers beside gates indicate the percentage of T1 (AA4.1+IgMhighCD232), T2 (AA4.1+IgMhighCD23+), and T3 (AA4.1+IgMlowCD23+) B cells (left). Summary of the frequencies of T1–T3 B cells (right). Each symbol represents a mouse. Data are shown as mean 6 Downloaded from SD. Statistical significance was deter- mined by one-way ANOVA (**p , 0.01, ***p , 0.001). http://www.jimmunol.org/

in vitro, and RNA harvested from the cultures was subjected to (PUMA) were expressed at higher levels in BAFF-stimulated RNA sequencing. nfkb2-deleted versus WT B cells (Supplemental Table I). To- Differentially expressed sequence analysis of NF-kB2– gether, these findings suggest that B cells with impaired signaling proficient versus NF-kB2–deficient BAFF-stimulated B cells iden- through the alternative pathway have an increased propensity to by guest on September 29, 2021 tified 150 genes with reduced and 174 genes with increased undergo apoptosis. In agreement, increased cell death of BAFF- (Supplemental Table I) expression in the absence of NF-kB2 at a stimulated B cells that express a RELB mutant protein that is significance threshold of padj , 0.05. The fold changes were unable to bind DNA compared with WT controls has previously mostly small, but highly significant. Genes were assigned to pu- been demonstrated (55). To assess the survival capacity of RELB/ tative functional categories (Supplemental Fig. 3B, Supplemental NF-kB2–deficient B cells in vitro, we stimulated B cells from Table I), including B cell markers, apoptosis, cellular trafficking, relbfl/flnfkb2fl/flCD19-Cre or CD19-Cre control mice with either and immune response T-dependent (Table I). A transcriptional BAFF or CD40 for 3 d, as both BAFF and CD40 are strong ac- profile analysis of BAFF-responsive genes controlled by the al- tivators of the alternative pathway. The results showed signifi- ternative NF-kB pathway has not previously been undertaken. cantly enhanced cell death in the cultures of RELB/NF-kB2– However, cd23 (encoding FcεRII) is a known BAFF-controlled deficient versus control B cells in response to BAFF stimulation gene in B cells (53), and the gene encoding ICOS ligand (∼63% versus ∼23% by propidium iodide [PI] staining and ∼21% (ICOSL) has been identified as a bona fide target of the alternative versus ∼66% by annexin V/7-AAD staining) (Fig. 6B, 6C). Of subunits in response to BAFFR stimulation in murine B cells (54). note, we also observed reduced expression of BAFFR on RELB/ In the RNA-seq analysis, we observed reduced expression of NF-kB2–deficient versus WT B cells (Fig. 6D), which may con- mRNA encoding cd23 and icosl in NF-kB2–deficient versus tribute to the observed enhanced cellular death. In contrast, cell control B cells (Table I). Accordingly, following BAFF or CD40 viability was comparable among the genotypes in response to stimulation, ICOSL protein was expressed at significantly reduced CD40 stimulation (cell death ∼14% versus ∼22% by PI staining levels on eGFP+ (i.e., relb/nfkb2-deleted) B cells derived from and ∼17% versus ∼20% by annexin V/7-AAD staining), although relbfl/flnfkb2fl/flCD19-Cre mice, whereas eGFP2 B cells from the the small difference observed in the PI staining reached signi- same mice displayed surface levels of ICOSL similar to those ficance (Fig. 6B, 6C). These findings suggest that CD40 stimu- observed on wild-type (WT) B cells (Fig. 6A). These results lation activates a transcriptional program in RELB/NF-kB2– validate our strategy for identifying BAFF-responsive targets. In deficient B cells that is able to overcome the viability defect the following sections, we present major findings of the RNA-seq observed in response to BAFF. In agreement with this finding, analysis. RNA-seq analysis of NF-kB2–deficient B cells and WT controls upon 6 h of CD40 stimulation in vitro revealed that there was little k Control of BAFF-mediated survival by the alternative NF- B overlap in the genes controlled by NF-kB2 following BAFF versus pathway CD40 stimulation (Supplemental Fig. 3C, Supplemental Table I). We observed a slightly reduced expression of mRNA encoding A Venn diagram demonstrates the overlap in genes with reduced the anti-apoptotic gene bcl2 in NF-kB2–deficient versus control expression in NF-kB2–deficient B cells upon BAFF versus CD40 B cells (Table I). In addition, the proapoptotic genes Bmf and Bbc3 stimulation (Supplemental Fig. 3D). Listed in Table I, we deter- The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Counterselection against relb/nfkb2-deleted follicular and MZ B cells in relbfl/flnfkb2fl/flCD19-Cre mice. (A) CD21 and CD23 expression of splenic B cells from mice of the indicated genotypes were analyzed by flow cytometry. Numbers beside gates indicate the percentage of follicular (CD23+ CD21int) and MZ (CD21highCD232) B cells (top). Summary of the frequencies of follicular and MZ B cells (bottom). Each symbol represents a mouse. Data are shown as mean 6 SD. Statistical significance was determined by one-way ANOVA (*p , 0.05, **p , 0.01, ***p , 0.001). (B) The fractions of eGFP+ cells among splenic follicular (CD23+CD21int) and MZ (CD21highCD232) B cells in relbfl/flnfkb2fl/flCD19-Cre mice were determined by flow cytometry. Numbers above gates indicate the percentage of eGFP+ B cells among the indicated B cell subsets (top). Summary of the frequency of eGFP+ cells among the corresponding B cell subsets (bottom). (C) Spleen sections from mice of the indicated genotypes were analyzed for the expression of eGFP and IgM by immunohistochemistry. FO, follicular area; MZ, marginal zone area. One representative mouse of three per group is shown. Original magnification 3400. Each symbol represents a mouse. Data are shown as mean 6 SD. Statistical significance was determined by Student t test (***p , 0.001). mined the expression of the genes that showed differential ex- hesion molecule L-selectin (Sell, CD62L), in NF-kB2–deficient pression following 6 h of BAFF stimulation with the expression B cells compared with controls following BAFF stimulation levels following 6 h of CD40 stimulation. Finally, genes were (Table I). These findings suggest that, in B cells, the alternative assigned to putative functional categories, and a pie chart sum- pathway controls a gene expression program that permits homing marizing these findings is shown in Supplemental Fig. 3C. to and positioning within B cell follicles in response to cues from stromal cells. We note that staining B cells from relbfl/flnfkb2fl/fl k Control of B cell trafficking by the alternative NF- B pathway CD19-Cre mice did not reveal significant differences in the ex- Both relb2/2 and nfkb22/2 mice show altered lymphoid organi- pression levels of CXCR5, CCR7, and CD62L (data not shown); zation (43–45), and analysis of total splenic mRNA revealed re- similarly, transwell migration assays using CXCL13, the ligand duced expression of the chemokines CXCL13, CCL19, and for CXCR5, on RELB/NF-kB2–deficient and WT B cells did not CCL21 compared with controls (46). Mildly reduced expression detect significant differences in the migration potential among the of the chemokine receptors CXCR5 and CCR7 was also observed genotypes (data not shown). We attribute these observations to the in splenic mRNA from relb2/2 mice (46). To our knowledge, possibility that the RELB/NF-kB2–deficient B cells that have these findings provided the first suggestion that the alternative reached maturity in relbfl/flnfkb2fl/flCD19-Cre mice may represent pathway may be required for proper lymphoid organization by a selected population of B cells that have survived and migrated to controlling the expression of chemokines or chemokine receptors the spleen and lodged in the follicles due to near normal expres- in stromal and/or other cell types. In support of this notion, RNA- sion of these genes. Future experiments aimed at inducibly de- seq analysis revealed reduced mRNA expression of the chemokine leting relb/nfkb2 specifically in B cells may help to clarify this receptors CXCR5, CCR7, and EBI2 (Gpr183), as well as the ad- matter. 8 CELL-INTRINSIC ROLES OF RELB AND NF-kB2 IN B CELLS

Table I. BAFF and CD40-responsive genes controlled by the alternative NF-kB pathway as identified by RNA-seq analysis of BAFF and CD40- stimulated nfkb2-deleted and WT B cells

6-h BAFF 6-h CD40

Functional Category Gene Fold Change padj Value Fold Change padj Value Nfkb2 22.53 3.72E-88 23.23 0 Immune response–B cell Fcer2a (CD23) 21.33 0.018 21.39 7.03E-58 Apoptosis Bcl2 21.33 0.029 Unchanged N.A. Trafficking Sell 21.47 1.49E-15 Unchanged N.A. Gpr183 (Ebi2) 21.49 1.27E-12 21.35 2.05E-20 Ccr7 21.61 3.95E-10 21.22 1.71E-12 Cxcr5 21.19 0.0099 Unchanged N.A. Immune response– H2-DMb2 21.32 8.73E-07 Unchanged N.A. T-Dependent Icosl 21.28 5.89E-06 Unchanged N.A. Ctss 21.27 0.00013 Unchanged N.A. H2-Oa 21.25 0.0038 Unchanged N.A. Cd83 21.92 0.0083 Unchanged N.A. H2-Aa 21.28 0.014 Unchanged N.A. N.A., not applicable. Downloaded from Because the absence of alternative NF-kB subunits correlated sides regulating ICOSL expression, the alternative NF-kB path- with a reduction in the fraction of MZ B cells (29, 46) (Fig. 5B, way may have a more substantial role in activating the expression 5C), we determined the surface protein expression of the integrin of genes required for optimal T cell–dependent B cell responses in chains aL and b2, which together form the LFA-1 complex, on response to BAFF. relb/nfkb2-deleted and WT control B cells. LFA-1 is important for

the retention of B cells within the splenic MZ (56). Whereas we Discussion http://www.jimmunol.org/ detected no significant differences in the expression level of aL BAFF-mediated activation of the alternative NF-kB pathway has between eGFP+ (i.e., relb/nfkb2-deleted) B cells from relbfl/fl long been implicated in the survival of mature B cells. However, nfkb2fl/flCD19-Cre and CD19-Cre control mice, relb/nfkb2-deleted the B cell–intrinsic roles of transcription factors downstream of B cells showed strongly reduced surface expression of b2 com- the pathway, RELB or NF-kB2, have not as yet been identified. pared with B cells from CD19-Cre control mice (Fig. 6E). This Perhaps most importantly, it is unknown how the combined de- observation is likely to be relevant to the strong counterselection letion of relb and nfkb2 affects the development and function of against eGFP+ MZ B cells observed in relbfl/flnfkb2fl/flCD19-Cre B cells. We here crossed newly generated conditional relb and mice (Fig. 5B, 5C), and may suggest an impaired retention of nfkb2 alleles, either separately or combined, to CD19-Cre mice to these cells in the MZ compartment (see Discussion). ablate these subunits specifically in B cells. We found that the by guest on September 29, 2021 CD21 has been identified as a target of BAFFR signaling (53). individual gene deletions led to a decrease in the fraction of We observed lower protein levels on the surface of eGFP+ relb/ mature B cells; however, the most dramatic reduction in peripheral nfkb2-deleted B cells in comparison with CD19-Cre control mice B cells was observed upon simultaneously ablating RELB and (Fig. 6F), suggesting that BAFF mediates expression of CD21 via NF-kB2. Thus, by ablating the downstream transcription factors of the alternative NF-kB pathway. High expression of CD21 is a the alternative NF-kB pathway in B cells, we define the extent to marker of MZ B cells (57). It is possible that reduced expression which this pathway regulates mature B cell homeostasis. of CD21 on RELB/NF-kB2–deficient MZ B cells impairs the Ag- Similar to what has been described in mice with B cell–con- stimulation properties of those cells as the CD19/CD21 coreceptor ditional deletion of a major regulator of the canonical pathway, complex has been implicated in lowering the threshold of BCR ikkb (60, 61), we observed a marked counterselection of relb/ signaling (58). nfkb2-deleted B cells in mice with B cell–specific RELB/NF-kB2 deficiency, which was strongest in the MZ B cell compartment. Genes involved in T cell/B cell interactions regulated by the Nevertheless, neither of these mouse models showed a complete k alternative NF- B pathway disappearance of ikkb or relb/nfkb2-deleted B cells, indicating that As mentioned above, the alternative NF-kB pathway has been the B cell phenotypes are less severe than those observed in mice implicated in the regulation of ICOSL expression on B cells. deficient in BCR or BAFF signaling that show a virtually complete ICOSL is involved in facilitating interactions with T cells, and absence of B cells. One possibility to explain these findings is that its expression on B cells is required for the development of the canonical and alternative NF-kB pathways may complement T-follicular helper cells (54). In addition to decreased expression each other to a certain extent during B cell development and of icosl in BAFF-stimulated NF-kB2–deficient versus control maintenance, as suggested by the observation that adoptive B cells, our RNA-seq analysis revealed reduced expression of transfer of bone marrow from nfkb12/2nfkb22/2 mice into RAG1- several genes involved in MHC class-II (MHCII) Ag presentation deficient animals resulted in the generation of only few mature (Table I). These included the genes ctss, H2-DMb2, and H2-Oa, B cells (62). It is, however, clear that non-NF-kB–mediated effects which respectively encode cathepsin S, H2-DMb2, and H2-Oa and of BAFFR or BCR stimulation also contribute to the survival of are involved in the processing and removal of invariant chain mature B cells (12–15, 63). peptide (CLIP) from MHCII molecules to allow loading of anti- In in vitro cultures, relb/nfkb2-deleted B cells were sensitive genic peptides. In addition, Cd83 was expressed at lower levels in to apoptosis when stimulated with BAFF, suggesting that the NF-kB2–deficient B cells compared with WT B cells (Table I). alternative NF-kB pathway controls an anti-apoptotic program in CD83 is a member of the Ig superfamily that is associated with normal B cells that is activated in response to BAFF stimulation. B cell activation, and CD83-deficient splenic B cells also have BAFF signaling has long been implicated in controlling B cell reduced MHCII Ag presentation (59). Thus, it appears that, be- survival (64), and, in agreement with this notion, ectopic expres- The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 6. Differential expression of BAFF-responsive genes controlled by the alternative NF-kB pathway. (A) Flow cytometric analysis of BAFF or CD40-stimulated B cells from relbfl/flnfkb2fl/flCD19-Cre and CD19-Cre mice at day 3 for the expression of ICOSL. Staining of eGFP+ and eGFP2 B cells from relbfl/flnfkb2fl/flCD19-Cre mice and B cells from CD19-Cre mice (top). Summary of the corresponding median fluorescence intensities (MFI) (bottom). eGFP+ and eGFP2 identify relb/nfkb2-deleted and nondeleted B cells from relbfl/flnfkb2fl/flCD19-Cre mice, respectively. Each symbol represents a mouse. Data are shown as mean 6 SD. Statistical significance was determined by one-way ANOVA (*p , 0.05, **p , 0.01, ***p , 0.001). (B) Flow cytometric analysis of BAFF or CD40-stimulated purified B cells from relbfl/flnfkb2fl/flCD19-Cre and CD19-Cre mice at day 3 for DNA content by PI staining (top) and fl/fl fl/fl summary of the corresponding percentage sub-G1 (bottom). (C) Flow cytometric analysis of BAFF or CD40-stimulated purified B cells from relb nfkb2 CD19-Cre and CD19-Cre mice at day 3 for apoptotic/dead cells by annexin V/7-AAD staining (top) and summary of the corresponding percentage of annexin V/7-AAD cells (bottom). (D) Flow cytometric analysis of ex vivo B cells from relbfl/flnfkb2fl/flCD19-Cre and CD19-Cre mice for BAFFR ex- pression (right) and summary of the corresponding MFIs (right). eGFP+ identifies relb/nfkb2-deleted B cells from relbfl/flnfkb2fl/flCD19-Cre mice. (E) Flow fl/fl fl/fl cytometric analysis of ex vivo B cells from relb nfkb2 CD19-Cre and CD19-Cre mice for b2 expression (left) and summary of the corresponding MFIs (right). eGFP+ identifies relb/nfkb2-deleted B cells from relbfl/flnfkb2fl/flCD19-Cre mice. (F) Flow cytometric analysis of ex vivo B cells from relbfl/flnfkb2fl/fl CD19-Cre and CD19-Cre mice for CD21 expression (left) and summary of the corresponding MFIs (right). eGFP+ identifies relb/nfkb2-deleted B cells from relbfl/flnfkb2fl/flCD19-Cre mice. (B–F) Each symbol represents a mouse. Data are shown as mean 6 SD. Statistical significance was determined by Student t test (*p , 0.05, **p , 0.01, ***p , 0.001). sion of BCL2 led to the accumulation of B cells in BAFFR- BAFF-responsive genes controlled by the alternative pathway, deficient mice (9, 65, 66). It is interesting to note that activation either directly or indirectly, that are involved in lymphocyte of relb/nfkb2-deleted B cells with CD40, also a strong inducer of trafficking, including adhesion molecules and chemokine recep- p100 processing (67), did not lead to increased cell death, indi- tors. Two previous studies have implicated the alternative pathway cating that CD40 signaling can transmit survival signals by a in the regulation of B cell homing to the lymphoid follicles and different route, presumably via the canonical NF-kB pathway (68). the MZ, respectively. Based on a RT-PCR analysis of mRNA de- Interestingly, RNA-seq analysis revealed limited overlap between rived from whole spleen of relb2/2 mice, Weih et al. (46) reported the genes controlled by NF-kB2 in response to BAFF versus CD40 slightly reduced mRNA levels of the chemokine receptors CXCR5 stimulation. This includes BCL2, whose mRNA expression changed and CCR7. Expression of CXCR5 is required for homing of slightly upon BAFF stimulation, whereas it remained unaffected B cells to the follicle, whereas CCR7 directs positioning at the B– upon CD40 stimulation. T cell boundary (69). We found both receptors to be expressed at BCL2 overexpression does not fully rescue the loss of B cells slightly lower mRNA levels in BAFF-stimulated NF-kB2–defi- in BAFFR-deficient mice (9, 65, 66), indicating that BAFF stim- cient B cells compared with controls. In addition, we observed ulation has additional functions besides providing prosurvival reduced expression of the chemokine receptor EBI2, which guides signals. In accordance, our RNA-seq analysis identified several B cell localization in the lymphoid microenvironment (70), and 10 CELL-INTRINSIC ROLES OF RELB AND NF-kB2 IN B CELLS the adhesion molecule L-selectin (CD62L), which mediates the References entry of lymphocytes from the blood into lymphoid tissue. These 1. Lam, K. P., R. Kuhn,€ and K. Rajewsky. 1997. In vivo ablation of surface im- results may suggest that RELB/NF-kB2–deficient B cells that fail munoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90: 1073–1083. to express chemokine receptors and adhesion molecules at phys- 2. Kraus, M., M. B. Alimzhanov, N. Rajewsky, and K. Rajewsky. 2004. Survival of iological levels may be impaired in their homing to the follicle. In resting mature B lymphocytes depends on BCR signaling via the Igalpha/beta agreement with this notion, mice with B cell–specific deletion of heterodimer. Cell 117: 787–800. 3. Mackay, F., and P. Schneider. 2009. Cracking the BAFF code. Nat. Rev. both relb and nfkb2 have fewer splenic B cells and fewer B cell Immunol. 9: 491–502. follicles. 4. Gross, J. A., S. R. Dillon, S. Mudri, J. Johnston, A. Littau, R. Roque, M. Rixon, k O. Schou, K. P. Foley, H. Haugen, et al. 2001. TACI-Ig neutralizes molecules A critical role for the alternative NF- B pathway in the devel- critical for B cell development and autoimmune disease: impaired B cell mat- opment of MZ B cells has been suggested based on two observa- uration in mice lacking BLyS. Immunity 15: 289–302. tions: First, relb2/2→WT chimeras show a significant reduction of 5. Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew, and M. L. Scott. 2001. An essential role for BAFF in the MZ B cells compared with the controls (46). Second, BAFF- normal development of B cells through a BCMA-independent pathway. Science transgenic mice and mice with inactivation of upstream compo- 293: 2111–2114. nents of the alternative pathway (that induce processing of p100), as 6. Thompson, J. S., S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, C. Hession, P. Schneider, I. D. Sizing, C. Mullen, et al. 2001. BAFF-R, a newly well as mice that lack p100 but still express p52, are characterized identified TNF receptor that specifically interacts with BAFF. Science 293: by MZ hyperplasia (10, 32–35, 71). As reported by Enzler et al. 2108–2111. 7. Yan, M., J. R. Brady, B. Chan, W. P. Lee, B. Hsu, S. Harless, M. Cancro, (29), the latter phenotype is most likely due to the upregulation of I. S. Grewal, and V. M. Dixit. 2001. Identification of a novel receptor for the integrin chains aLb2 (LFA-1) and a4b1 (VLA-4) that facilitate B lymphocyte stimulator that is mutated in a mouse strain with severe B cell homing to the MZ (56). The expression of these genes was found to deficiency. Curr. 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Functions of NF-kappaB1 and NF-kappaB2 in in mature B cell homeostasis. The constitutive activation of these immune cell biology. Biochem. J. 382: 393–409. transcription factors may promote cell survival and distort traf- 23. He, J. Q., S. K. Saha, J. R. Kang, B. Zarnegar, and G. Cheng. 2007. Specificity of TRAF3 in its negative regulation of the noncanonical NF-kappa B pathway. J. ficking within the lymphoid tissue, and potentially lead to sus- Biol. Chem. 282: 3688–3694. tained activation of B cells, which may contribute to disease 24. Sun, S. C. 2012. The noncanonical NF-kB pathway. Immunol. Rev. 246: 125– pathogenesis. 140. 25. Claudio, E., K. Brown, S. Park, H. Wang, and U. Siebenlist. 2002. BAFF- induced NEMO-independent processing of NF-kappa B2 in maturing B cells. Acknowledgments Nat. Immunol. 3: 958–965. 26. Kayagaki, N., M. Yan, D. Seshasayee, H. Wang, W. Lee, D. M. French, We thank Thomas Ludwig and Evangelos Pefanis for advice on the targeting I. S. Grewal, A. G. Cochran, N. C. Gordon, J. Yin, et al. 2002. BAFF/BLyS vectors and the members of the Klein laboratory for insights and discus- receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface sion. We also thank Victor Lin of the Transgenic Mouse Shared Resource loop and promotes processing of NF-kappaB2. Immunity 17: 515–524. and the Molecular Pathology and Flow Cytometry Shared Resources of 27. Morrison, M. D., W. Reiley, M. Zhang, and S. C. Sun. 2005. An atypical tumor necrosis factor (TNF) receptor-associated factor-binding motif of B cell- the Herbert Irving Comprehensive Cancer Center. activating factor belonging to the TNF family (BAFF) receptor mediates in- duction of the noncanonical NF-kappaB signaling pathway. J. Biol. Chem. 280: 10018–10024. Disclosures 28. Hatada, E. N., R. K. Do, A. Orlofsky, H. C. Liou, M. Prystowsky, The authors have no financial conflicts of interest. I. C. MacLennan, J. Caamano, and S. Chen-Kiang. 2003. NF-kappa B1 p50 is The Journal of Immunology 11

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B Supplementary Figure 1. Generation of conditional relb and nfkb2 alleles. (A) Relb (RELB) targeting strategy (for experimental details, see Supplementary Methods below) (top). Shown from top to bottom i) the relb locus with the promoter region, exons and translational start site in exon 1 (E1); ii) the targeting vector comprising an eGFP, a triple SV40 polyA site (tpA), a PGK neopA cassette, a PGK promoter (P) downstream of exon 2, a TK gene, and loxP and frt sites; arrows denote the orientation; iii) the targeted relb locus and iv) the locus after Cre-mediated recombi- nation of loxP sites. Restriction sites, probes used for detection and the expected fragments detected by Southern blot analysis are indicated. (Bottom) Correctly targeted embryonic stem (ES) cell lines were identified by Southern blot analysis of EcoRV-digested DNA that displayed the 5.9 kb band of the integrated transgene along with the 11.4 kb wild-type (WT) band. Co-inte- gration of the 3’ loxP site was verified by the presence of the expected 4.8 kb transgenic band. (B) Nfkb2 (NF-kB2) targeting strategy (for experimental details, see Supplementary Methods below) (top). Shown from top to bottom i) the nfkb2 locus with the promoter region, exons and translational start site in exon 2 (E2); ii) the targeting vector comprising an eGFP, a tpA site, a PGK neopA cassette, a PGK promoter (P) downstream of exon 2, a TK gene, and loxP and frt sites; arrows denote the orientation; iii) the targeted nfkb2 locus and iv) the locus after Cre-mediated recombination of loxP sites. Restriction sites, probes used for detection and the expected fragments detected by Southern blot analysis are indicated. (Bottom) Correctly targeted ES cell lines were identified by Southern blot analysis of PvuII-digested DNA that displayed the 8.3 kb band of the integrated transgene along with the 5.2 kb WT band. Co-integration of the 3’ loxP site was verified by PCR analysis using primers that hybridize in a unique region spanning the PGK promoter and the 3’ frt site (forward primer) and in a region upstream of intron 3 of nfkb2. A B C

*** ***

* * *

eGFP+

(millions of cells) of (millions cells) of (millions cells) of (millions

Absolute # splenic Bcells splenic Absolute# Bcells splenic Absolute# Bcells splenic Absolute#

relbfl/fl nfkb2fl/fl

CD19-Cre CD19-Cre CD19-Cre

Supplementary Figure 2. Reduced numbers of splenic B-cells in the absence of alternative NF-kB subunits. (A-C) Absolute numbers of splenic B-cells from relbfl/flCD19-Cre, nfkb2fl/flCD19-Cre or relbfl/flnfkb2fl/flCD19-Cre mice and the corresponding heterozygous and CD19-Cre control mice. Data are shown as mean ± standard deviation. Statistical significance was determined by Student’s t test (*, P<0.05; ***, P<0.001). A BAFF D 6h BAFF 6h CD40

p100/p52

pAKT total AKT β-actin padj<0.01 B C

6h BAFF; padj<0.05 6h CD40; padj<0.01 Supplementary Figure 3. Identification of BAFF and CD40-responsive genes controlled by the alternative NF-kB pathway by RNA-seq analysis of BAFF and CD40-stimulated nfkb2-deleted and WT B-cells. (A) Western blot analysis of purified WT B-cells ex vivo (d0), after 3h culture in medium only, and stimulated for 6h and 24h with BAFF. (B) Differentially expressed sequence (DE-SEQ) analysis of NF-kB2-proficient vs. NF-kB2-deficient BAFF-stimulated B-cells identified 150 genes with reduced expression in the absence of NF-kB2 at a significance threshold of padj<0.05. Genes were assigned to putative functional categories. For the identity of the corresponding genes, fold-change and padj values, see Supplementary Table I. (C) Differentially expressed sequence analysis (DE-SEQ) of NF-kB2-proficient vs. NF-kB2-deficient CD40-stimulated B-cells identified 306 genes with reduced expression in the absence of NF-kB2 at a significance threshold of padj<0.01. Genes were assigned to putative functional categories. For the identity of the corresponding genes, fold-change and padj values, see Supplementary Table II. (D) Venn diagram depicting the overlap of genes that showed reduced expression in the absence of NF-kB2 upon both BAFF and CD40 stimulation at a significance threshold of padj<0.01. For the identity of the corresponding genes, fold-change and padj values, see Supplementary Table III. Supplementary Table I 6h BAFF Stimulation DOWN LIST FUNCTIONAL CATEGORY GENE FOLD CHANGE padj value ANTI-APOTOTIC Bcl2 1.325543529 0.02945239 CYTOKINE Il16 1.174198435 0.015844786 Il12a 1.364670688 0.025448272 CYTOSKELETON Psd 2.710416969 3.31E-50 Reep3 1.413310723 5.22E-08 Ccdc99 1.639556254 0.000630591 Dynlt3 1.28487809 0.000651276 Tuba1b 1.310452963 0.013300371 Tubb5 1.183371423 0.016273855 Lamb3 1.457520181 0.006795493 DNA REPAIR Bre 1.254578558 0.005088337 Neil1 1.231318507 0.011716652 Lig1 1.266981951 0.041050674 IMMUNE RESPONSE - B CELL Fcer2a (CD23) 1.325732056 0.017623548 IMMUNE RESPONSE - INNATE Tmem173 1.494749042 2.57E-15 Irak3 1.331124105 0.000166854 Ticam2 1.605212254 0.001376318 Map3k8 1.324359846 0.001711831 Gbp5 1.311710295 0.003818353 Lrrc33 1.211129325 0.006204436 IMMUNE RESPONSE - T-dependent H2-DMb2 1.315573363 8.73E-07 H2-Oa 1.249701592 0.003787994 Icosl 1.279887924 5.89E-06 Ctss 1.267684422 0.000133023 Cd83 1.192126349 0.008264942 H2-Aa 1.279103195 0.014128882 METABOLISM Chst10 1.646260235 1.42E-20 B4galnt1 1.342557059 3.86E-08 Pgap1 1.414539953 1.20E-06 Ugcg 1.280810535 2.33E-05 Prdx6 1.316376842 9.93E-05 Faah 1.664599634 0.000220923 Sdhaf1 1.332327141 0.001090296 Naaa 1.35377873 0.001711831 Kynu 1.285099975 0.003828254 Alkbh6 1.257097276 0.013127776 Hsd17b11 1.234976001 0.016890233 Oat 1.219954321 0.018450351 Sgms1 1.2008314 0.025188693 Kmo 1.173450616 0.040117349 OTHER Lpcat1 1.679481466 1.39E-18 Syngr2 1.368374306 7.05E-10 Gpr132 1.418653018 4.29E-09 P2ry10 1.416996618 1.04E-07 Gpr174 1.351249124 1.05E-07 Srgn 1.344730774 2.31E-07 Stk17b 1.284992045 1.31E-06 Tmem123 1.266805908 7.34E-06 Prcp 1.322126349 7.34E-06 Cnp 1.352586628 3.02E-05 Ppp1r16b 1.28170883 0.000139812 Panx1 1.369383341 0.000220731 Prss12 1.6583446 0.000562756 Smap2 1.222848398 0.000762389 Filip1l 1.223693318 0.000850199 Zfp36l1 1.217865092 0.000910526 Fam69a 1.26830035 0.002361236 Bmp2k 1.264286133 0.006693482 Smagp 1.457531848 0.010647072 Ttpal 1.191433902 0.012988025 Fam111a 1.248348619 0.013757943 Rdm1 1.304869816 0.018049232 Sesn1 1.184896876 0.01922234 Tgif1 1.182398998 0.025188693 Plek 1.241242607 0.028269871 Cytip 1.1701271 0.029403356 Scn8a 2.147250624 0.030652721 Csrp1 1.307827093 0.031092574 Zdhhc18 1.167138677 0.031345903 Tmem8 1.26459962 0.036128199 Hmgn1 1.186759232 0.036523669 Hpse 1.33345198 0.038481626 Snx8 1.167525968 0.04194061 Gtpbp1 1.184101515 0.047488424 Ms4a4c 1.412947428 3.99E-10 Rbpms 1.440213131 0.028769288 PROLIFERATION Mcm5 1.374548993 6.03E-06 Pola1 1.436176686 0.011209285 Cables1 1.44033209 0.027286066 Mcm2 1.239161005 0.03791968 RIBOSOME Rps15a 1.371883866 8.30E-05 Rps24 1.222974504 0.000685617 Rpl32 1.284748379 0.000845661 Rps15 1.272048419 0.001263385 Rps3a 1.219342319 0.001961946 Rps5 1.315793758 0.001987104 Rpsa 1.27300757 0.002992526 Rps17 1.327365118 0.003865064 Rps11 1.198842614 0.003865064 Rpl21 1.313838181 0.006942322 Rps25 1.299329208 0.009376262 Rps20 1.301480356 0.017351649 Rps18 1.284337556 0.017623548 Rplp1 1.25196849 0.019607973 Rps4x 1.242016261 0.03328255 Rpl22 1.27621509 0.040756683 RNA Snrnp25 1.531122318 1.31E-11 Dhx57 1.246873827 0.000629503 Dhx40 1.204614275 0.018834808 Znrd1 1.275235877 0.03328255 Ddx25 1.687054793 0.041050674 SIGNALLING Stat4 1.479017416 1.60E-14 Tifa 1.405759008 8.02E-07 Cyth1 1.27202071 2.77E-05 Pde4b 1.268933849 4.17E-05 Rapgef1 1.255319991 5.03E-05 Grm6 1.626442754 5.90E-05 Sfn 1.280482713 7.70E-05 Rasgef1b 1.284530883 0.000419028 Ppp3ca 1.21579356 0.006090489 Mapk11 1.293431975 0.0064556 Gng10 1.296843924 0.006871586 Stat6 1.182310174 0.011645639 Dennd4a 1.266048973 0.016890233 Gpr18 1.269401893 0.019567736 Mapk12 1.378862817 0.021083551 Grap 1.272861725 0.025740469 Gbp4 1.218677047 0.027353694 Rras2 1.193438225 0.030652721 Rasa3 1.169639635 0.035721026 Syk 1.154232114 0.04580206 Pik3cd 1.183538141 0.012281839 TRAFFICKING Sell 1.471424122 1.49E-15 Gpr183 (Ebi2) 1.491272082 1.27E-12 Cxcr5 1.185785738 0.009919532 Ccr7 1.609781954 3.95E-10 TRANSCRIPTION Pou2af1 (OCA-B) 1.376137691 3.62E-11 Ets2 1.517653974 6.08E-07 Trim24 1.242155151 0.008863833 Spic 1.64995705 0.017174723 Ets1 1.163928278 0.022668769 TRANSPORT Abcc1 1.361944309 6.92E-07 Slc20a1 1.265175303 0.000132885 Slc2a3 1.256328465 0.013127776 UNKNOWN Rnf157 1.439456186 5.40E-08 Fam169b 1.304064186 1.70E-06 1300014I06Rik 1.434671289 8.30E-05 I730030J21Rik 1.660975421 0.001727066 Kbtbd11 1.273509517 0.002091503 Arrdc3 1.247105275 0.002091503 Zfp958 1.345445679 0.004537295 Fam107b 1.193584764 0.004563295 6230427J02Rik 1.795926088 0.005214902 15-Sep 1.236788766 0.008944745 Pqlc1 1.274168472 0.012594903 Ly6a 1.214844256 0.013580634 Crisp3 1.385696438 0.016312525 Zfp608 1.212734532 0.020015354 Zmat2 1.200039092 0.032788432 Mir682 1.316900195 0.037378097 Nfkb2 2.534237408 3.72E-88

UP LIST FUNCTIONAL CATEGORY GENE FOLD CHANGE padj value APOPTOSIS Bmf 1.65972061 7.24E-18 Bbc3 (PUMA) 1.602398362 0.002756872 B-CELL-SPECIFIC Fcrl5 (IRTA2) 1.55685032 7.61E-07 CYTOKINES Il10 3.968080839 0.000229336 Il12b 3.256747297 0.027089435 CYTOSKELETON Myo1g 1.39419205 8.88E-10 Ccdc88b 1.877179269 1.96E-09 Vim 1.530263792 8.73E-07 Ssh3 1.389174726 0.000716074 Ppl 1.791479862 0.002556438 Tubb2b 1.636812511 0.003624001 Sspn 1.956492802 0.004133041 Epb4.1l2 1.24688001 0.005611603 Vasp 1.202044796 0.008882303 Lrrc16a 1.289955402 0.010647072 Col11a2 1.544285219 0.030448929 DNA REPAIR Polm 1.446524167 0.004511346 IC TRAFFICKING Cplx2 2.310715937 1.63E-11 Stxbp1 1.854395487 1.99E-09 Ehd2 1.520200238 1.29E-05 Itsn1 1.380361577 0.001792806 IMMUNE CELL-specific Cd93 1.718718609 5.09E-08 Rag1 2.611717576 0.009856109 Cd44 1.304220533 0.000845661 IMMUNE RESPONSE - Ag pres H2-Q4 1.18160423 0.027089435 Lilrb3 1.266567534 0.031177751 H2-T24 1.551205223 0.000522479 Lrmp 1.556443325 0.001917319 IMMUNE RESPONSE - INNATE Tlr12 1.85666738 0.016883121 METABOLISM Mgll 3.306638136 1.12E-06 Scd2 1.337186265 3.14E-06 Pld4 1.555203766 3.60E-06 Pfkfb3 1.335451739 0.000522938 Serinc5 1.53084655 0.000562756 Gad1 1.379001693 0.000651276 Adssl1 1.524260273 0.003865064 Gns 1.185790081 0.015916005 Ephx1 1.196044916 0.021921341 Aldh3b1 1.408427183 0.022668769 Man2a2 1.211480894 0.04837014 Padi2 1.698509786 0.002091503 OTHER Myof 1.97537815 1.12E-06 Ski 1.342692476 4.51E-06 Cacna1e 2.180152448 1.30E-05 Cd36 1.614495279 3.52E-05 Ceacam1 1.484156469 6.23E-05 P2rx7 2.522779678 0.000139812 Psap 1.229355515 0.000280845 Sema4b 1.276660723 0.00028676 Chst3 1.236295424 0.000341418 Akap12 1.628510684 0.000373941 Mfhas1 1.551160076 0.0004705 Ermap 2.108700363 0.000486286 Slfn5 1.39007718 0.000716191 Bhlhe41 2.211166293 0.000721515 Sbk1 1.250090186 0.000796129 Ddit4 1.548451661 0.000850199 Anxa6 1.299845861 0.001601099 Cacna1h 2.216175621 0.002091503 Pitpnm2 1.328399418 0.00250882 Chrnb1 1.482908083 0.003501811 Bcl7a 1.219872147 0.008189727 Plac8 1.201274585 0.008189727 Capn2 1.341838252 0.008884271 Ankrd13b 2.826201546 0.010647072 Midn 1.22986858 0.012988025 Gramd4 1.207224294 0.015141179 Pea15a 1.311059957 0.015546891 Leprotl1 1.317667921 0.017623548 Ldlrap1 1.265427008 0.020044034 Fam126a 1.258812468 0.020078026 Tmc6 1.202302472 0.022160316 Nrp2 2.966739515 0.025568096 Ret 1.718560278 0.025821009 Vwf 3.14323754 0.02843997 Plau 4.506826964 0.029247347 Mreg 1.336153784 0.031177751 Txndc5 1.228409254 0.031370276 Vangl2 1.813726173 0.031858297 Ankrd13d 1.365886221 0.03414065 Entpd1 1.297766431 0.03414065 Mgst1 1.63195404 0.041148798 Trio 1.370346122 0.041388395 Cd300lf 2.021806619 0.047199353 Sorcs2 1.943011395 0.047488424 Gpr56 2.224602872 5.46E-05 Ndrg1 2.078787078 8.90E-05 Alpl 1.618002936 0.012281839 Csda 1.251669977 0.012623083 PROLIFERATION Cdkn1a 1.917659738 0.015844786 PROT DEG Nedd4 1.779161016 1.36E-06 Asb2 2.426063337 6.86E-06 Znrf3 1.921626815 0.020078026 Spsb1 2.342476744 0.040268915 PROT STAB Hspa1a (Hsp70-1) 5.695433618 0.003575741 Hspa1b (Hsp70-1b) 5.653293421 0.003785318 Hsph1 (Hsp105) 1.394889811 0.006204436 RNA Akap17b 1.657912082 0.003192827 Zfp385a 1.696129503 0.046728948 SIGNALING Csf2rb 1.685757509 0.007818925 Hck 1.427269462 0.044854085 Sh2d3c 1.20554822 0.049821404 Wnt10a 2.270625494 5.98E-06 Ptprs 1.419476773 2.02E-05 Pde4a 1.54521352 8.30E-05 Pde4c 3.584870496 0.00032063 Prex1 1.259246403 0.000850199 Rgs16 2.409556442 0.001130216 Nfkbid (ikbdelta) 1.321156627 0.001442443 Rasal1 1.357421117 0.001987104 Prkcc 2.41388695 0.004511346 Mcf2l 3.12026425 0.009778409 Tbc1d9 2.015899948 0.010133594 Notch1 1.452631168 0.011023733 Camk2n1 1.70615537 0.013314237 Sbf2 1.428543916 0.01631898 Rasgrp1 1.186950546 0.023282283 Card11 1.202173451 0.027089435 Fgd2 1.415367791 0.03214068 Rgs2 1.413159148 0.035292485 Ptger4 1.31817624 0.035657449 Arhgap22 3.17048857 0.047917149 Gadd45b 1.274776673 0.013314237 Pik3c2b 1.292350717 0.0363481 Dusp10 1.466706856 0.037795143 Inppl1 1.303723978 0.039449574 TRAFFICKING Plxnd1 1.67076192 5.36E-09 S1pr3 2.007543312 0.010647072 Ccr1 1.846591874 0.017231401 TRANSCRIPTION Hivep3 2.401702315 1.74E-10 E2f2 1.517881193 4.29E-09 Egr1 1.572770367 3.95E-06 Cbx6 1.489462934 4.16E-06 Myb 1.882719151 5.34E-06 Hdac9 1.419631399 5.54E-05 Pou2f2 (Oct-2) 1.285403536 7.28E-05 Zbtb32 1.70449426 0.000850199 Tle3 1.447015761 0.006796405 Klf10 1.457623253 0.013314237 Id2 2.012132476 0.01631898 Mafg 1.557442984 0.017555605 Egr3 1.617609745 0.017623548 Arid3a 1.272936652 0.020970289 Tfeb 1.248220816 0.041050674 Egr2 1.366652935 0.047277274 TRANSPORT Atp2a3 1.30898498 6.08E-07 Atp1b1 1.927424598 4.24E-05 Slco4a1 1.83499065 0.001045242 Slc12a4 1.477917824 0.001711831 Abcb4 1.771772685 0.009726483 Slc7a7 1.589285148 0.010647072 Sfxn3 1.220610506 0.013830368 Slc29a3 1.271817491 0.023865009 Orai2 1.231216397 0.02619518 UNKNOWN Fam167a 1.543222633 4.69E-08 E330020D12Rik 1.611926172 4.19E-07 Myadm 1.914808864 1.12E-06 11-Sep 1.363877503 6.86E-06 Rapgefl1 2.86823894 8.30E-05 Ccdc123 1.377680746 0.000950196 Heatr6 1.27480024 0.002091503 2700081O15Rik 1.504156524 0.003865064 Fam53b 1.281283446 0.004176585 Ahdc1 1.31907436 0.008189727 Emid1 1.676734373 0.009376262 4930506M07Rik 2.981996994 0.013314237 Lrrc32 2.803024746 0.01634561 Tm6sf1 1.384744771 0.016890233 Cobll1 1.281385277 0.016890233 Zfp395 1.265067085 0.017623548 Gm6307 2.902272473 0.023301272 Zcchc24 1.332098962 0.040756683 2210403K04Rik 1.592064594 0.041161547 Efr3b 6.413563779 0.04136992 Endod1 1.326848769 0.043648057 6h CD40 Stimulation DOWN LIST FUNCTIONAL CATEGORY GENE FOLD CHANGE padj value ANTI-APOTOTIC Bcl2l1 (BclXL) 1.34155305 6.32E-12 Pim3 1.253142978 0.000516377 Bcl2a1d (BFL1) 1.169640061 0.001951387 Bcl2a1b 1.147470384 0.00486127 CYTOSKELETON Psd 3.789653827 3.68E-208 Mid1 2.348629878 1.43E-87 Tubb5 1.169641523 5.04E-11 Reep3 1.207771701 2.85E-07 Tuba4a 1.194137281 4.84E-05 Dynll2 1.188647277 0.000362681 Ccdc99 1.296359809 0.000363293 Cdc42se2 1.128547667 0.000789093 Dclk2 1.418036458 0.003411005 Marcks 1.231155296 1.31E-17 Marcksl1 1.141840852 0.00110408 DNA REPAIR Usp1 1.186931822 0.000120426 Lig1 1.242697852 0.004429219 Parp1 1.089669158 0.005017591 IMMUNE RESPONSE Cd53 1.346704277 4.27E-31 Ly9 1.216497679 5.98E-12 Il2ra 1.190811053 2.16E-07 Tlr1 1.200520448 2.28E-07 Cd48 1.193560168 6.16E-06 Il17ra 1.195159776 1.24E-05 Slamf1 1.382172464 0.000155919 Cd82 1.143054647 0.000178597 Cd37 1.096998251 0.002449181 Icam1 1.108308489 0.002482829 H13 1.133010567 0.003006722 Sema7a 1.10384842 0.008295074 Btla 1.391990203 7.45E-27 Lta 1.456963397 2.28E-07 Trim59 1.318295114 1.17E-06 Itgal (CD11a, LFA1A) 1.108177234 0.001235358 Irgm1 1.178228362 0.001850091 Lsp1 1.079452556 0.008634966 IMMUNE RESPONSE - B CELL Fcer2a (CD23) 1.393769543 7.03E-58 Cr2 (CD21) 1.470272525 8.26E-35 Irf4 1.214311635 8.91E-16 Cd19 1.141225132 9.12E-08 Pax5 1.138510353 0.000395993 Ibtk 1.127853001 0.002389566 Ebi3 1.474930946 2.60E-12 Mef2c 1.183028777 3.39E-09 Bank1 1.189354268 3.97E-09 Ms4a1 (CD20) 1.130739935 4.17E-07 Bcl6 1.197540041 6.35E-06 IMMUNE RESPONSE - INNATE Ticam2 1.545643331 2.96E-21 Lrrc33 1.273320878 2.63E-12 Gbp3 1.188107444 4.51E-05 Ifi47 1.180806635 0.002070411 IMMUNE RESPONSE - T DEPENDENT Cd83 1.135022947 1.63E-07 INTRACELLULAR TRAFFICKING Hip1 1.519144103 9.12E-08 M6pr 1.113375844 0.002620666 INTRACEULLAR TRANSPORT Abcf2 1.13352646 0.006370451 METABOLISM B4galnt1 1.287222005 4.01E-21 Aacs 1.271616781 2.18E-13 Acly 1.18616871 2.54E-10 Fasn 1.205765752 4.00E-09 Pgap1 1.320987712 8.59E-09 Lss 1.245118802 5.42E-08 Ugcg 1.19272406 6.79E-08 Slc25a1 1.279050159 3.60E-07 Sqle 1.166759521 1.18E-06 Dhcr24 1.204787417 2.06E-06 Pank3 1.213635315 1.07E-05 Cblb 1.190450274 1.36E-05 Mthfd1l 1.163762135 8.18E-05 Pcyt2 1.15276139 0.00112152 Ndufaf4 1.190308723 0.003037615 Hk2 1.239836728 0.004028234 Mid1ip1 1.214107828 0.00481226 Acsl3 1.202306124 0.005467203 Qdpr 1.16670527 0.005958451 Sc4mol 1.1376175 0.007171286 Slc4a7 1.141395652 0.0071949 Gpd2 1.211740715 0.008349758 Idi1 1.245585583 0.000232616 Ppat 1.14855941 0.005847866 Srebf2 1.093568546 0.002273002 Stard4 1.207021596 1.08E-05 Insig1 1.333952993 1.85E-18 Ldlr 1.248104324 1.89E-14 Atp1a1 1.109986215 0.000880047 Cyp51 1.178212631 1.26E-08 Slc1a5 1.110170102 0.000899093 Slc16a1 1.216050082 0.002793183 Srm 1.148689716 0.004047777 Atad3a 1.124050162 0.00844028 Shmt2 1.093789097 0.008845554 St6gal1 1.229621391 1.29E-10 Hmgcr 1.170612571 3.80E-08 Atic 1.105013004 0.008109975 Elovl6 1.297217248 0.00560321 NUCLEAR STRUCTURE Tmpo 1.157109218 8.15E-06 Nol8 1.150696065 0.000799011 Ipo5 1.10168242 0.002779277 Cse1l 1.102901274 0.00722962 MISCELLANEOUS Tmem123 1.266529926 6.70E-28 Gpr174 1.474861908 2.56E-26 Filip1l 1.328642752 5.40E-26 Fam111a 1.304434838 8.05E-20 Zfp36l1 1.253347985 1.87E-19 Plek 1.223762947 1.61E-15 Ankrd33b 1.282620617 8.19E-13 Ms4a4c 1.288022567 7.34E-12 Nolc1 1.201106779 3.55E-09 Agrn 1.560665124 3.59E-09 Gnl3 1.201238655 2.24E-08 Setbp1 1.575917532 3.97E-08 Fam113b 1.274461765 4.16E-07 Lpcat1 1.210780237 2.29E-06 Lmo2 1.39345804 4.39E-06 Stk17b 1.153207203 1.33E-05 Pglyrp2 1.402290393 3.54E-05 Txnrd3 1.339736058 4.00E-05 Ttpal 1.150642697 9.08E-05 Zfp62 1.132947943 0.000101338 Gtpbp1 1.146802292 0.000255132 Napsa 1.123141886 0.000809536 Bzw2 1.166087379 0.000860197 Fen1 1.181113411 0.00102801 Scand1 1.409083649 0.001049016 Nr6a1 1.474206834 0.001767978 Smagp 1.688609969 0.001998791 Sema5a 1.572149974 0.002209321 Bmp2k 1.127457001 0.006745201 Cnbp 1.08098495 0.009788553 Ier5 1.197467409 1.62E-08 Nup62 1.208929974 5.10E-07 Phtf2 1.211884881 1.59E-06 Pprc1 1.164152228 6.35E-06 Klf3 1.2940379 1.41E-05 Arl6ip1 1.115261666 0.002623894 Tmem63b 1.156167174 0.007211629 Atl3 1.110456162 0.008109975 F9 1.588175522 0.008833272 Chchd10 1.26542279 0.002712511 Dek 1.12625915 0.00117945 Zfp296 1.262017636 0.00158867 Ifrd2 1.160273822 0.004009587 PROLIFERATION Ccnd2 1.288480065 2.17E-20 Mcm5 1.220002199 3.71E-07 Mir17hg 1.213884366 9.19E-05 Ddx11 1.283109532 0.001862058 Recql4 1.528323952 0.003779339 Mcm7 1.19577173 0.004522672 Nasp 1.137913769 0.005561035 Cdca7 1.184595209 0.004700159 Anp32b 1.117078981 0.006745201 Cdk5r1 1.400366388 1.12E-12 Smc1a 1.130881573 0.000756511 PROTEIN DEGRADATION Ubqln4 1.180898547 0.000145922 Wsb2 1.251118214 0.002155822 Dcaf12 1.171311175 0.003430123 Hspa5 1.095879776 0.000517767 Usp36 1.156884639 0.000649964 Rad18 1.298612704 0.001994037 Josd1 1.166783501 0.002245763 Fbxl15 1.659832283 0.005025488 PROTEIN STABILITY Cct5 1.093219025 0.003195919 Hspa8 1.102380071 0.00458051 RIBOSOME Rpl11 3.523389174 1.24E-24 Ncl 1.147214925 9.93E-10 Heatr1 1.167835451 2.24E-08 Nop56 1.168221138 1.28E-06 Nop58 1.148831787 5.87E-06 Tsr1 1.173929293 2.00E-05 Ftsj3 1.137047335 4.78E-05 Pa2g4 1.117257089 0.000152028 Syncrip 1.114632547 0.00027331 Imp4 1.184290908 0.000370096 Mphosph10 1.168665368 0.000415641 Rpl10 1.222444418 0.001185294 Rpl10a 1.200114567 0.00136193 Ubtf 1.115534025 0.001392164 Dhx33 1.143170522 0.002912482 Rrp12 1.134782405 0.003289913 Nop14 1.1365709 0.003896434 Urb2 1.15080925 0.005852759 Bop1 1.130269587 0.006112593 Rrs1 1.14910155 0.008179197 Rsl1d1 1.104756862 0.009486006 Gtpbp4 1.126157291 0.002273002 Sdad1 1.106366631 0.0086791 Cirh1a 1.139733149 0.006737376 Nop2 1.126860238 0.002035004 Nol9 1.16032377 0.000224712 Dkc1 1.130505247 0.00047357 Hnrnpu 1.097745642 0.00047357 Rpl7 1.106152733 0.00061473 Hnrnpa0 1.159311068 0.001365163 RNA Dhx40 1.270793382 2.40E-07 Srsf2 1.147819483 2.44E-07 Ppan 1.200797143 5.47E-07 Srsf7 1.127512017 5.39E-05 Serbp1 1.108273385 0.000157448 G3bp1 1.119559942 0.00017884 Dhx57 1.11849215 0.002332114 Srsf6 1.098862665 0.002654044 Sf3b3 1.093285538 0.003881138 Abce1 1.114150147 0.000363622 Snora81 1.468408072 0.006885024 Polr1e 1.171529406 0.004792319 SIGNALLING Slamf7 1.245492048 3.37E-12 Rasgef1b 1.448687138 4.00E-20 Plxnc1 1.271641428 2.27E-16 Grm6 1.508137189 3.05E-12 Rapgef6 1.228984857 7.54E-10 Grap 1.157526527 6.44E-09 Csrnp1 1.24710214 4.42E-08 Map2k1 1.187413108 1.22E-07 Nfkbia (ikBalpha) 1.142707394 4.32E-06 Stat6 1.134310896 7.04E-06 Pde1c 3.731136065 8.02E-05 Pik3cd 1.122694866 0.000436945 Ppp3ca 1.169492356 0.000525809 Pde4b 1.122619211 0.007682164 Evc 10.75185085 5.78E-07 Ccdc50 1.138865085 1.58E-05 Dennd4b 1.11220639 0.000876391 Gadd45g 1.243984744 0.003289913 Vav3 1.368669625 0.003741905 Pde3b 1.133024739 0.005390185 Fgd2 1.158299384 0.009921965 Lfng 1.172722571 0.009981245 Pikfyve 1.256992215 2.20E-06 Bcl9 1.184078727 0.0008383 Ttyh3 1.219798136 5.37E-07 Map4k1 1.114860406 0.001035185 Rhof 1.219714727 2.51E-08 Arap2 1.17865052 5.50E-06 St8sia6 1.465244638 2.12E-17 Fam116b 1.39688589 3.46E-09 Gripap1 1.213470841 3.51E-09 Stk4 1.098111541 0.002066848 Birc2 1.123452904 0.002901661 Tnfaip3 (A20) 1.093738624 0.008139919 Crtc2 1.247076428 1.57E-11 TRAFFICKING Gpr183 1.349485578 2.05E-20 Ccr7 1.218279376 1.71E-12 TRANSCRIPTION Nr1d2 1.254806994 0.000284761 Rel 1.201492873 0.000285129 Jun 1.280293374 0.00040487 Arid3b 1.197725719 0.000702309 Lef1 1.332847469 0.003430123 Casz1 1.229952198 0.004665717 Papd5 1.154311175 0.006487049 Taf4b 1.192343859 0.007790175 Polr1b 1.136986108 0.001898839 Foxp4 1.154634549 1.98E-05 Zbtb10 1.191556857 0.000720357 Mybbp1a 1.085470735 0.005376771 Clock 1.178813079 0.006094422 Mybl1 1.755820372 0.009981245 Myc 1.247502695 1.69E-10 Ddx21 1.15454876 2.87E-08 Jund 1.205432866 0.001303148 TRANSCRIPTION - EPIGENETICS Phf15 1.382438039 3.59E-34 Kdm6a 1.19636632 0.000116843 Kdm6b 1.179093862 0.000129925 Naa50 1.126687757 0.000330363 Phf12 1.138716788 0.001198829 Jarid2 1.120999712 0.001403393 Set 1.139146987 0.001994037 Cbx7 1.138882932 0.002248993 Nat10 1.115835231 0.005432258 Ddx3x 1.140102556 5.18E-07 Sfmbt1 1.222033661 0.000697238 Kdm5c 1.144155095 0.008751749 TRANSLATION Eif3a 1.102245337 0.000686623 Eif3b 1.107263452 0.000985055 Eif1a 1.124484371 0.004463689 Tars 1.140498256 0.005690737 Naa25 1.148504255 0.005702513 Eif4a2 1.149765203 2.25E-08 Icmt 1.159986168 0.002174136 Nsun2 1.097567496 0.007122281 Iars 1.102806075 0.008981367 Wdr4 1.147616847 0.009125164 Mat2a 1.1418767 6.04E-06 Eif2s3x 1.389847849 0.000112582 UNKNOWN Fchsd2 1.266173811 4.09E-15 Fam129c 1.157671448 5.14E-09 G530011O06Rik 1.93799725 5.84E-09 1300014I06Rik 1.534920437 6.22E-09 Fam55b 1.701222835 2.28E-07 Wdr43 1.160509193 2.29E-06 2410002F23Rik 1.194249639 6.84E-06 Tmem64 1.2556279 2.47E-05 1300001I01Rik 1.151457425 2.72E-05 2210021J22Rik 1.656976506 0.000129874 Znfx1 1.144560653 0.000940133 Gm7120 1.668877088 0.003112492 4930413G21Rik 1.484695311 0.004139412 Wdr75 1.129243836 0.005852759 Khnyn 1.11058595 0.007275303 Zfp771 1.451819553 0.008631025 Fam107b 1.279472962 1.17E-24 E130012A19Rik 1.421589113 0.004665717 Cebpz 1.112523042 0.005901536 Zbtb11 1.151073547 0.005950478 Pwp2 1.124079112 0.006972359 Pus7 1.144837575 0.004665717 Qser1 1.163675702 0.006016401 Supplementary Table 1. Identification of BAFF or CD40-responsive genes controlled by the alternative NF-B pathway by RNA-seq analysis of BAFF or CD40-stimulated nfkb2- deleted and WT B-cells. Differentially expressed sequence analysis (DE-SEQ) of NF-B2- proficient vs. NF-B2-deficient BAFF or CD40-stimulated B-cells (6 hours) identified genes with altered expression in the absence of NF-B2. Genes were assigned to putative functional categories. Fold-change and padj values are indicated.