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Impairment of Mature B Cell Maintenance Upon Combined Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 is online at: average * The Journal of Immunology published online 5 February 2016 from submission to initial decision B Transcription Factors RELB and NF- κ B2 in B Cells 4 weeks from acceptance to publication http://www.jimmunol.org/content/early/2016/02/04/jimmun ol.1501120 κ Nilushi S. De Silva, Kathryn Silva, Michael M. Anderson, Govind Bhagat and Ulf Klein J Immunol Impairment of Mature B Cell Maintenance upon Combined Deletion of the Alternative NF- Submit online. Every submission reviewed by practicing scientists ? is published twice each month by http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://www.jimmunol.org/content/suppl/2016/02/04/jimmunol.150112 0.DCSupplemental Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material Permissions Email Alerts Subscription Supplementary The Journal of Immunology 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. This information is current as of September 29, 2021. 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.
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