Mary Kaileh                                                      NF-jB function in B lymphocytesRanjan Sen                ...
Kaileh & Sen Æ NF-jB function in B lymphocytesB lymphopoiesis occurs in the fetal liver and provides the neo-             ...
Kaileh & Sen Æ NF-jB function in B lymphocytesresponsive to the survival cytokine BAFF (also known as BLyS)       producti...
Kaileh & Sen Æ NF-jB function in B lymphocytes   Normal recombination order produces pro-B cells that                   me...
Kaileh & Sen Æ NF-jB function in B lymphocytesRestoration of k+ cells by ectopic expression of a Bcl-2 trans-      by extr...
Kaileh & Sen Æ NF-jB function in B lymphocytesFig. 2. Simplified scheme of peripheral B-cell differentiation. Transitional ...
Kaileh & Sen Æ NF-jB function in B lymphocytesMZ B cells, and their status in RelA deficiency has yet to beclearly defined. ...
Kaileh & Sen Æ NF-jB function in B lymphocytesnuclear levels of classical NF-jB proteins, such as RELA, but             NF...
Kaileh & Sen Æ NF-jB function in B lymphocytesshuttling of NF-jB proteins between the nucleus and                     sign...
Kaileh & Sen Æ NF-jB function in B lymphocytesmediated by the kinase PDK1 which, like Btk, contains a PH                au...
Kaileh & Sen Æ NF-jB function in B lymphocytesscenario, where there is limited BCR engagement with antigen,       CsA poin...
Kaileh & Sen Æ NF-jB function in B lymphocytes   Despite persistent induction of RELA, Rel-deficient B cells do          LP...
Kaileh & Sen Æ NF-jB function in B lymphocytesstimuli to NF-jB. Identification of the components involved                  ...
Kaileh & Sen Æ NF-jB function in B lymphocytes       determine transcriptional activity and          26. Grawunder U, Haas...
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  1. 1. Mary Kaileh NF-jB function in B lymphocytesRanjan Sen Summary: NF-jB proteins were identified in the search for mecha-Authors’ address nisms that regulate B-lymphocyte-specific transcription of immunoglob-Mary Kaileh1, Ranjan Sen1 ulin j light chain genes. Twenty-five years later, though the function of1 Gene Regulation Section, Laboratory of Molecular Biology the jB site in the enhancer remains enigmatic, NF-jB proteins have beenand Immunology, National Institute on Aging, National Insti- shown to have important roles in B-cell development, maintenance, andtutes of Health, Baltimore, MD, USA. function. In this review, we summarize the functions of NF-jB in B cells. An overview of B-cell biology that identifies stages in the life of B lym-Correspondence to: phocytes for the general reader is followed by three sections that examineRanjan Sen the role of NF-jB family of proteins in B-cell development, mature B-cellGene Regulation Section survival and B-cell function. We endeavor throughout to suggest mecha-Laboratory of Molecular Biology and Immunology nisms and implications of the wide-ranging observations that have beenNational Institute on Aging made and conclude by highlighting the need to understand NF-jB-medi-National Institutes of Health ated gene expression in more depth.251 Bayview BoulevardBaltimore, MD 21224, USA Keywords: NF-jB, B lymphocytes, BCR, function, developmentTel.: +410 558 8630Fax: +410 558 8386e-mail: ranjan.sen@nih.govAcknowledgements Overview of B-cell biologyThe authors are supported entirely by the Intramural The adaptive immune system is characterized by the ability toResearch Program of the NIH, National Institute on Aging(Baltimore, MD). The authors have no conflicts of interest to respond to pathogens that have never been encountereddeclare. before, to gear the response to the type of pathogen and to retain memory of the pathogenic encounter for subsequent challenge. This program is manifested by B and T lymphocytes This article is part of a series of of the immune system and the specially designed antigen reviews covering NF-jB appearing in receptors that are expressed by these cells. Unlike cells of the Volume 246 of Immunological Reviews. innate system that recognize limited numbers of pathogen- Video podcast available associated molecular patterns, each B and T lymphocyte Go to expresses a unique antigen receptor. Thereby, lymphocytes to provide millions of potential antigen-recognition specificities, watch interview with Guest Editor and generation of a diverse repertoire of lymphocytes is a key Sankar Ghosh feature of lymphoid development. To mount an effective immune response against any given pathogen, a limited num- ber of recognition specificities are selected from this vast rep- ertoire to be amplified and further differentiated to provide protection. At the end of the response a subset of pathogen-Immunological Reviews 2012 specific cells must be preserved to provide memory. Thus, theVol. 246: 254–271 history of lymphocytes consists of development, survival for aPrinted in Singapore. All rights reserved finite time period, selection by antigens for expansion andPublished 2012. This article is a US Government work function, and long-term survival of memory.and is in the public domain in the USAImmunological Reviews For B cells, development starts with hematopoietic stem0105-2896 cells present in the fetal liver or the adult bone marrow. Fetal Published 2012. This article is a US Government work and is in the public domain in the USA254 Immunological Reviews 246/2012
  2. 2. Kaileh & Sen Æ NF-jB function in B lymphocytesB lymphopoiesis occurs in the fetal liver and provides the neo- which leads to replacement of the light chain gene variablenate with B cells. The functionality of these cells remains an region (5, 6). Association of the new light chain with thearea of much study, however it is very likely that they provide existing IgH chain may, or may not, change receptor speci-a degree of protection soon after birth (1). For much of the ficity away from self-reactivity. If it does, recombinationlifetime of vertebrates, B lymphopoiesis occurs in the adult ceases and the immature B cell can migrate to the periph-bone marrow. A difference between B lymphocytes produced ery. Immature B-cells which remain self-reactive despitein the fetal liver and the bone marrow is the specificity of their consecutive VL replacements are eliminated by apoptosis. Ofantigen receptor repertoires. Generation of antigen receptor the approximately 18 million immature B cells that are pro-diversity occurs at the distinct steps of B-cell development via duced daily in mice, it has been estimated that approxi-stage-specific rearrangement of immunoglobulin (Ig) heavy mately 1–2 million migrate to peripheral lymphoid organs,(H) and light (L) chains genes (2–4). IgH gene assembly takes such as the spleen. It seems plausible that some form ofplace first in pro-B cells (Fig. 1). Because V(D)J recombination selection determines which immature B cells leave the boneis error-prone, not all cells that initiate IgH gene rearrange- marrow.ments successfully produce IgH protein. IgH+ and IgH) cells Immature B cells that reach the spleen undergo further dif-are distinguished on the basis of signals transduced via a pre- ferentiation to produce mature B cells that are competent toB-cell receptor (pre-BCR) composed of IgH chains and light- mount immune responses (7, 8). Two fundamental changeschain-like molecules k5 and V-pre-B. pre-BCR+ cells undergo accompany peripheral B-cell differentiation. First, the func-several rounds of cell division to produce pre-B-cells where tional consequences of BCR crosslinking are altered. NewlyIgL rearrangements take place. arrived immature B cells in the spleen, like their counterparts Light chains come in two varieties, kappa (j) and lambda in the bone marrow, die in response to BCR crosslinking. This(k), in mice and humans. In pre-B cells, Igj rearrangements allows immature B cells to ‘checkout’ the peripheral environ-occur first, followed by Igk rearrangements. Since all pre-B ment for potential self-reactivity. An important differencecells contain IgH proteins, successful production Igj or Igk between antigen recognition by immature B cells in the spleenallows IgH ⁄ L-containing B-cell receptors (BCRs) to be versus the bone marrow is that receptor editing does notexpressed on the cell surface. These cells are called immature occur in the spleen. During transition from immature toB cells. If the BCR on the surface of an immature B-cell in the mature B cells, the response to BCR-crosslinking changes frombone marrow recognizes self-antigen, receptor editing occurs, cell death to cell proliferation. Second, mature B cells becomeFig. 1. Simplified scheme of B-cell differentiation in the bone marrow. Stages of B-cell differentiation from hematopoietic stem cells (HCS) dis-cussed in this review are highlighted. The state of immunoglobulin (Ig) heavy (H) and light (L) chain gene rearrangements are noted below eachstage. The pre-B-cell receptor is composed of IgH chains and surrogate light chains; immature B cells express B-cell receptors composed of IgH plusIgL. Receptor editing occurs in response to BCR cross-linking on immature B cells in the bone marrow, with the objective of reducing self-reactivityby altering antigen recognition specificity. Cells that continue to be self-reactive are deleted by apoptosis, while those that are not can be exported tothe periphery.Published 2012. This article is a US Government work and is in the public domain in the USAImmunological Reviews 246/2012 255
  3. 3. Kaileh & Sen Æ NF-jB function in B lymphocytesresponsive to the survival cytokine BAFF (also known as BLyS) production by p50 ⁄ RelA double-deficient precursors is causedwhich is essential for B-cell homeostasis. by a similar systemic stress response. While the simplest inter- Peripheral differentiation produces two major mature B- pretation of the synergistic effect of the double-deletion is thatcell subsets: (i) mature marginal zone (MZ) B cells that can p50 and RELA work together as NF-jB, it is also possible thatmount T-dependent and T-independent immune responses, alternative functions of the p105 precursor are involved inand (ii) follicular (FO) B cells that are the major source of promoting cell survival during differentiation. Finally, InlayT-dependent immunity and B-cell memory. In addition, a et al. (22) performed the key experiment of generating micethird subset of mature B cells, known as B1 B cells, are with mutations in the jB site of ijE. Igj rearrangement, de-present in the periphery; these are believed to be of fetal methylation and expression was unchanged on the mutantorigin (8). Here we review the role of NF-jB proteins in B- allele, thereby providing incontrovertible evidence that thecell development and function. Because this area of research ijE jB site was not essential for j gene expression during nor-has been reviewed before (9, 10), we highlight the possi- mal B-cell development.ble mechanisms involved and interesting interpretations ofthe data, with a goal towards identifying areas for futurestudy. Possible functions of the j enhancer jB site The strong conservation of the ijE jB site across species (23) suggests that it serves an important function. One possibility NF-jB proteins in B-cell development is that the ijE jB site regulates the developmental timing ofNF-jB proteins were identified based on binding to a highly the j gene rearrangements in the bone marrow. During differ-conserved sequence referred to as the jB site within the j entiation, IgH gene rearrangements occur first at the pro-B celllight chain gene enhancer (ijE) (11). Presence of NF-jB DNA stage followed by Igj (or k) rearrangements at the subsequentbinding activity only in B lineage cells that transcribed IgL pre-B-cell stage. Functionally, the order of rearrangementsgenes, the demonstration that the jB element was important ensures that each IgH chain that is generated pairs with multi-for transcriptional activity of ijE (12) and later evidence of ple light chains to increase B-cell repertoire diversity. How-the role of ijE in Igj gene rearrangements (13, 14) suggested ever, recent studies show that the j locus is in a permissivea role for NF-jB transcription factor in j gene activation dur- nuclear environment for recombination at the pro-B-cell stageing B-cell development. The first indication that this was not (24, 25); yet, j transcription and the bulk of j recombinationtrue came from the analysis of Nfkb1 gene deleted mice, which is restricted to the pre-B-cell stage. Taken together with thelacked the p50 component of the ‘classical’ p50 ⁄ p65 NF-jB observation of low levels of j recombination in pro-B cellsheterodimer (15–17). B-cell development and j gene expres- (26, 27), one possibility is that p50 homodimer binding tosion was unaffected in these mice (18) calling into question the jB site may suppress recombination in pro-B cells. Reliefthe prevailing hypothesis. Subsequently, p65 (RelA)-defi- of suppression in pre-B cells would permit robust recombina-ciency was also shown to not affect B-cell development (19). tion to occur. In this scenario, mutation of the jB site is The double-deficiency of p50 and RelA was examined by predicted to simply increase the level of j rearrangement inreconstituting fetal liver cells of the appropriate genotypes pro-B-cells. Since IgH rearrangements would occur simulta-into irradiated mice (20). In these experiments p50 ⁄ RelA pre- neously, such a timing defect may not be reflected in grosslycursors generated B cells, but only in the presence of wild type disrupted B-cell development. Rather, alteration of the ordercells. While demonstrating that p50 ⁄ RelA NF-jB was not of rearrangements could be reflected more significantly inessential for B-cell development (and by inference, Igj rear- the diversity of the B-cell repertoire that is generated. Alongrangements), these observations also pointed towards an the same conceptual lines, the ijE jB site may fine tune jessential cell non-autonomous NF-jB-dependent function gene expression in mature B cells. For example, p50 homo-provided by hematopoietic cells for B-cell development. Based dimers bound the jB site could reduce j gene transcription inon a very similar phonotype of IjB kinase 2 (IKK2)-deficient resting B cells by recruiting histone de-acetylases that areprecursors, Karin and colleagues proposed that systemic TNFa associated with gene repression (28). During inflammatoryproduced during hematopoeitic reconstitution killed off T cell responses, p50 homo-dimers would be replaced by transcrip-precursors that were unable to induce NF-jB (21). This effect tion activating p50 ⁄ p65 or p50 ⁄ Rel heterodimers to enhancewas reduced when wild-type cells differentiated in the j gene expression required to secrete large amounts of anti-same milieu as the mutant cells. Perhaps the lack of B-cell bodies of defined specificity. Published 2012. This article is a US Government work and is in the public domain in the USA256 Immunological Reviews 246/2012
  4. 4. Kaileh & Sen Æ NF-jB function in B lymphocytes Normal recombination order produces pro-B cells that ments. It is interesting to recall that onset of cellular quies-express IgH chains but not IgL chains. Association of IgH cence has been related to induction of NF-jB and j genechain with non-rearranging k5 and V-pre-B polypeptides rearrangements in cell culture models as well. Specifically,(surrogate light chains) expressed in pro-B cells generates a Abelson virus transformed pro- ⁄ pre-B-cell lines contain verysignaling competent pre-B-cell receptor (pre-BCR). The pre- little nuclear NF-jB and do not express Igj. Induction of qui-BCR rescues pro-B cells from programmed cell death, induces escence by turning off the Abl oncogene results in NF-jBproliferation and differentiation to the pre-B-cell stage. The induction and j gene expression and rearrangementssignificant similarities in signaling pathways initiated at the (46–48). In these systems, blocking NF-jB induction reducespre-BCR and the BCR (29, 30) have suggested that the pre- j transcription and rearrangements. Another possible functionBCR activates NF-jB during the pro-B to pre-B-cell transition. for pre-BCR induced NF-jB could be to induce chemokineIndeed, use of NF-jB reporter mice (31) as well as direct and chemokine receptor expression that would permit pre-DNA binding assays (32) demonstrate higher levels of BCR-expressing cells to migrate to a different microenviron-transcriptionally competent NF-jB in pre-B cells compared to ment within the bone marrow. This may, in part, explain thepro-B cells. Recently a role for ATM, downstream recombina- reduced numbers of pre- and pro-B cells in p100) ⁄ ) andtion-induced DNA double strand breaks, has been proposed as p50) ⁄ RelB) mice (49, 50). Because such functions of pre-an NF-jB-inducing stimulus (33). How such a signal coordi- BCR induced NF-jB are likely to enhance rather than play annates with the pre-BCR-dependent developmental cues essential role in development, they may not be obvious inremains unclear. steady-state analysis of bone marrow sub-compartments. Yet, there is little genetic evidence for an important devel- Alternatively, NF-jB functions at these stages may be mani-opmental role for NF-jB in the bone marrow. Other than the fested at times of inflammatory stress or immune deficiencyp50 ⁄ RelA-double deficiency described above, none of the sin- when accelerated development would be or compound mutations of NF-jB proteins result in a dis-cernible block in B-cell differentiation (9, 10, 34, 35). Nor,do conditional deletion of the IjB kinases affect the numbers NF-jB function in immature B cells in the bone marrowor properties of pro- and pre-B cells (32, 36–38), despite Successful completion of light chain gene rearrangementsreducing NF-jB DNA binding activity in pre-B-cells. Interest- terminates V(D)J recombination at IgL loci and producesingly, modulating IjB function has been shown to affect early immature B cells that express immunoglobulin (BCR) on theB-cell differentiation. Specifically, ectopic expression of non- cell surface. Cells expressing BCRs that are crosslinked by self-degradable IjBa blocks transition of CD43+ pre-B cells to antigens in the bone marrow (self-reactive BCRs) undergoCD43) pre-B-cells (31, 39), and the double-deletion of IjBa receptor editing (Fig. 1). This process attempts to alter BCRand IjBe leads to substantial loss of pre- and pro-B cells in the specificity by re-activating V(D)J recombination at IgL loci tobone marrow (40). The increased apoptosis observed in pre-B generate new IgL chains to pair with the pre-existing IgHcells that express a non-degradable form of IjBa suggests an chain. Because Igk rearrangements follow the cessation of Igjanti-apoptotic role for pre-BCR-induced NF-jB, which can be rearrangements (51, 52), a substantial proportion of Igk-rescued by transgenic provision of Bcl-xL. There is evidence expressing B cells in the mouse are produced as a consequencethat apoptosis in pro-B cells that lack effective IjB function of receptor editing. Several observations are consistent with amay be mediated by TNFa (41). role for NF-jB proteins at this stage of differentiation. First, Despite the lack of obvious developmental defects in B lym- NF-jB DNA binding activity can be detected in immaturephopoiesis, it is tempting to speculate on the possible roles of B cells, particularly those that are engaged in receptor editingpre-BCR-induced NF-jB besides survival. First, the genes IRF4 (53, 54). Second, conditional deletion of NEMO, the double-and 8 have been shown to be essential for appropriate devel- deletion of IKK1 and IKK2, or deletion of TRAF6 (32) resultsopment to pre-B cells (42–44). IRF4) ⁄ IRF8) pre-BCR+ cells in a lower frequency of k+ immature B cells. Third, double-continue to divide in response to IL-7 and do not initiate j deletion of Nfkb1 and 2 genes leads to reduced numbers ofgene rearrangements. Because IRF-4 has been proposed to be immature B cells (55).a NF-jB target gene (45), it may be one of the targets of Analysis of the NEMO-deleted mice showed that the reduc-pre-BCR-induced NF-jB. In this way pre-BCR signaling would tion in k+ cells was not a consequence of reduced receptorinitiate a self-limiting proliferative phase; cessation of cell editing because recombinase (RAG) gene expression anddivision would permit the activation of j gene rearrange- RAG-induced DNA breaks were unaffected in this strain (32).Published 2012. This article is a US Government work and is in the public domain in the USAImmunological Reviews 246/2012 257
  5. 5. Kaileh & Sen Æ NF-jB function in B lymphocytesRestoration of k+ cells by ectopic expression of a Bcl-2 trans- by extreme susceptibility to apoptosis upon BCR stimulation,gene suggested a role for NF-jB-dependent survival; however, making them the target of negative selection against self-reac-Bcl-xL expression was not affected in NEMO or IKK1 ⁄ 2-defi- tivity in the periphery. BCR-induced cell death is mediated bycient immature B cells. Instead, mRNA of another pro-survival the mitochondrial pathway utilizing the pro-apoptotic BH3-kinase, Pim2, was attenuated in the absence of IKK activity. only domain proteins Bak, Bax, and Bim (61–63). T1 cellsPim2 is considered to be a target of the noncanonical (alter- develop into mature B cells via an intermediate transitional-2nate) NF-jB pathway (56) and implicated in the survival of (T2) stage. Whether all T1 cells that arrive in the spleen differ-mature B cells in response to BAFF (57, 58). Thus, extended entiate further is not clear. Indeed, a current view is that T1survival during receptor editing may be mediated, in part, by cells are destined to die unless ‘positively selected’ for furtherBAFF-dependent mechanisms. It is also possible that other differentiation (64, 65).receptors and ligands activate the noncanonical NF-jB path- A critical role for NF-jB proteins during T1 to T2 differentia-way at this differentiation step. A model that incorporates tion is evident from several genetic mutations (Fig. 2). First,both canonical (classical) and noncanonical NF-jB pathways Rel ⁄ RelA double-mutant fetal liver cells produce only T1 cellswould explain the requirement for p50 ⁄ p52 in immature after transfer to irradiated hosts (66, 67). The extreme sensitiv-B cells as well as the role of TRAF6 in generating of k+ cells. ity to apoptosis of the residual IgM+ cells in this genotype isConsistent with a dual signaling model, recent studies show partially rescued by a Bcl2 transgene; however, differentiationthat immature B cells, particularly those that express self-reac- remains incomplete indicating that Rel proteins are requiredtive sIg, are BAFF-responsive in vitro (59). for differentiation as well as cell survival. Consistent with the Overall, the following model emerges from these observa- idea that REL and RELA are required for differentiation to T2tions. Immature B cells in the bone marrow that express cells, conditional deletion of either NEMO or the double dele-self-reactive BCRs activate classical NF-jB via a NEMO ⁄ IKK- tion of IKK1 and IKK2, also blocks differentiation to T2 cellsdependent pathway. One of the consequences is up regulation (32). These observations suggest that signal-induced activationof BAFF-receptor on these cells, making them more responsive of classical NF-jB pathway proteins is essential for T2 differen-to BAFF-dependent survival signals. Extended survival of these tiation. The fact that single deletion of either Rel or RelA, orcells permits continual receptor editing that is required to either IKK gene, does not significantly impair differentiationgenerate k+ immature B cells. Disruption of canonical NF-jB likely reflects compensatory activity of the remaining proteins.activation could affect cellular longevity by making cells less The most likely source of NF-jB-induction in T1 cells is thesensitive to BAFF or by reducing levels of p100, an NF-jB tar- BCR. This is most clearly exemplified by conditional deletionget gene that serves as the substrate for the alternate NF-jB of CD79a, the BCR signal transducing module, in immaturepathway. As proposed by Derudders et al. (32), such short- B cells in vitro (68, 69). Absence of this protein leads to dra-lived cells would initiate receptor editing but not be able to matic alteration of the gene expression profile in these cellsproceed to ‘all the way’ to generate normal numbers of k+ towards a pattern that is more similar to that seen in pro-Bimmature B cells. Despite the requirement for NEMO or cells. Thus, constitutive BCR signaling maintains the state ofIKK1 ⁄ 2, the signaling pathway from the BCR to NF-jB in T1 cells and is required for developmental progression. Addi-immature B cells remains unclear. In particular, k+ cell numbers tionally, deficiency of several cytoplasmic signaling moleculesare unaffected in Bcl10-deficient mice (32, 60), leading to the that transduce BCR signals in mature cells also affect periph-proposal that the BCR on immature B cells may signal to IKKs by eral differentiation at transitional stages. The most prominenta CBM-independent pathway. Additional studies are required to among these are Btk, PLCc, PI3K and the adapter proteinsclarify how the BCR activates NEMO ⁄ IKK in these cells. BLNK and BCAP; however, the effect of each mutation varies (70–79). Thus, the case for BCR signaling in transitional B cells is strong. NF-jB function in peripheral B-cell differentiation However, the case for BCR signaling to NF-jB is relativelyImmature B cells that arrive in the spleen are referred to as weak. The strongest evidence against this idea is that defi-transitional cells and require further differentiation into ciency in any of the CBM complex (Carma-1, Bcl10, andmature functional B cells (7, 8) (Fig. 2). The most immature Malt1) proteins does not affect peripheral B-cell differentiationtransitional-1 (T1) cells are virtually indistinguishable from significantly (80–84). Because these proteins are essential forimmature B cells in the bone marrow with regard to function BCR-induced NF-jB activation in mature B and T cells (85,and expression of cell surface markers. They are characterized 86), the lack of developmental phenotypes of CBM mutations Published 2012. This article is a US Government work and is in the public domain in the USA258 Immunological Reviews 246/2012
  6. 6. Kaileh & Sen Æ NF-jB function in B lymphocytesFig. 2. Simplified scheme of peripheral B-cell differentiation. Transitional Type 1 (T1) cells are the most immature cells to arrive in the spleen fromthe bone marrow. T1 cells differentiate via an intermediate T2 stage to mature follicular (FO) and marginal zone (MZ) B cells. A T3 stage has been pro-posed but is not discussed in this review. Single- or compound gene deletions that affect peripheral differentiation are noted and discussed in the text.has been taken to indicate that BCR signaling to NF-jB is not signaling (see below). Third, T2 cells begin express higherinvolved in transitional B cells. There is some evidence, how- levels of BAFF-R and become responsive to survival signalingever, that absence of Bcl10 adversely affects maturation of T2 by BAFF. Two observations provide clues as to how BAFF-Rcells to follicular B cells (60). Moreover, biochemical studies expression is regulated in the T1 to T2 transition: (i) BCRalso indicate that BCR crosslinking by anti-Ig does not induce crosslinking upregulates BAFF-R expression in mature B cellsclassical NF-jB DNA binding in T1 cells (87, 88). How can (93), and (ii) this does not occur in Rel-deficient cells (88).we reconcile the requirement for inducible canonical NF-jB Thus, upregulation of BAFF-R in T2 cells may also reflect thecomponents in T1 ⁄ T2 differentiation with the apparent connection of BCR signaling to Rel in these cells. Overall, T2absence of a connection to the BCR? The most prosaic expla- cells bear a ‘more competent’ BCR and higher levels of BAFF-nation is that the NF-jB-inducing signal originates at a recep- R, which together may determine many of the characteristicstor other than the BCR. While we cannot rule out this of these cells.possibility till such a receptor is identified, we consider it While development beyond T2 stage requires BAFF ⁄ BAFF-Rmore likely that the BCR in T1 cells is connected to IKKs dif- interactions, recent evidence indicates that such signals mayferently than it is in mature cells (89). Mechanistically, this be initiated earlier in T1 cells. Specifically, Hoek et al. (94)may be because the BCR in T1 cells is not associated with lipid showed that B-cell development in mice deficient for both Btkrafts (90–92), resulting in a distinct constellation of signaling and BAFF-R was blocked at the T1 development stage. Sinceproteins in its vicinity. each individual mutation blocks differentiation at the T2 Differentiation to the T2 transitional stage requires alternate stage, these observations suggested that some BAFF signalingNF-jB function. This is exemplified by blocks at the T2 stage occurred at the earlier stage as well. Additionally, Rowlandof development in Nfkb1 and Nfkb2 double-deficient precursors et al. (59) showed that differentiation of immature bone mar-(55), as well as in IKK1-deficient precursors (56). The row B cells to transitional cells was enhanced in the presenceresponse of T2 cells to BCR crosslinking is distinct from T1 of BAFF (T1 cells express BAFF-R, though at lower levels thancells in several ways that involve NF-jB proteins. First, BCR T2 cells). Perhaps the reduced importance of BAFF ⁄ BAFF-R atcrosslinking induces classical NF-jB and long-term REL induc- the T1 stage is in part due to inefficient BCR signalingtion in T2 cells (88). Both these are characteristic of the NF- that prevents BAFF-R upregulation or efficient generation ofjB response of mature B cells (see below). Consequently, REL genes, such as Bcl-xL and A1, are activated in T2 cellsbut not in T1 cells. This is likely to be an important mecha-nism that makes T2 cells less susceptible to BCR-induced cell NF-jB proteins in mature B-cell survivaldeath. The molecular basis for differential BCR signaling to NF-jB proteins have essential roles in the generation and ⁄ orREL in T2 cells is not known. Second, BCR signals in T2 cells maintenance of mature B cells. Of the two major subsets of Bupregulate expression of Nfkb2 gene resulting in the produc- cells produced in the adult, MZ B cells are more susceptible totion of p100 protein (88). This is potentially an important NF-jB deficiency. This is reflected in reduced MZ B cellsaspect of the survival characteristics of T2 cells because p100 numbers in several Rel-family single gene deficiencies, includ-is an important substrate for BAFF-R-dependent survival ing Nfkb1 and RelB (Fig. 2). Absence of Rel appears not to affectPublished 2012. This article is a US Government work and is in the public domain in the USAImmunological Reviews 246/2012 259
  7. 7. Kaileh & Sen Æ NF-jB function in B lymphocytesMZ B cells, and their status in RelA deficiency has yet to beclearly defined. Follicular (FO) mature B cells are less affected BAFF ⁄ BAFF-R activates canonical and noncanonical NF-jBby single gene deficiencies, though they are significantlyreduced in several compound deficiencies such as Nfkb1 ⁄ 2, BAFF ⁄ BAFF-R interaction activates multiple pro-survivalNfkb1 ⁄ RelB, and RelA ⁄ Rel. Both kinds of mature B-cell subsets mechanisms in mature B cells, including activation of the non-are also reduced in mice that lack IKK1 and IKK2 or NEMO. canonical NF-jB pathway (103, 104). In accordance with anThese observations demonstrate that both canonical and important role for this pathway in B-cell survival, Nfkb2-defi-noncanonical pathway NF-jB proteins are required for mature cient B cells are refractory to BAFF-dependent survival in vitro.B-cell generation and ⁄ or maintenance, and that their function However, the presence of mature follicular B cells inmust be induced via the IKKs. Nfkb2- and RelB-deficient mice (105, 106) indicates that other Two receptors that are known to be essential for mature B- features of BAFF-R signals compensate for the lack of thesecell survival are the BCR and the BAFF-R. Mice that carry proteins in vivo. One of the prominent nuclear targets of thegenetic mutations in genes encoding either BAFF or the BAFF- resulting p52 ⁄ RelB heterodimer is the gene encoding theR lack mature B cells (95, 96). BAFF ⁄ BAFF-R stimulation is Ser ⁄ Thr kinase Pim2 (56). Unlike Nfkb2-deficient B cells, how-required continuously to maintain the peripheral B-cell pool ever, Pim2-deficient B cells respond to BAFF treatment in vitrosince intravenous administration in mice of Fc receptor fusion with increased viability indicating that p52 ⁄ RelB must activateproteins that bind BAFF results in the rapid loss of mature B additional survival genes in BAFF-treated cells (58). Thesecells (97, 98). The survival response of mature B cells to BAFF currently unknown genes appear to be downstream of mTORrequires a signaling-competent BCR. This was shown by con- because BAFF-responsiveness of Pim2-deficient cells is abro-ditional deletion of genes encoding either the immunoglobu- gated by rapamycin. Woodland et al. (58) connected BAFF-lin heavy chain (99) or the signal-transducing chaperones Iga dependent survival signaling via mTOR and PIM2 to theand Igb (100); disruption of the BCR complex led to loss of induction ⁄ maintenance of Mcl-1 expression. Though themature B cells within 24–48 h, despite the presence of sys- mechanism of Mcl-1 induction ⁄ maintenance is not clear, thistemic BAFF. is an important connection because, along with components Because the BCR on naive B cells provides survival signals in of the BCR or BAFF-R and BAFF, Mcl-1 is essential for survivalthe absence of overt antigen, this kind of signaling has been of mature B cells in mice (107).referred to as ‘tonic signaling’ (101). BCR signaling that In addition to the noncanonical NF-jB pathway, BAFF ⁄occurs in immature B cells (discussed above) is also a kind of BAFF-R interaction has also been shown to activate canonicaltonic signaling, though its similarity to survival signaling in NF-jB via a Btk-dependent pathway, resulting in induction ofmature B cells remains unclear. While the term tonic signaling RELA- and REL-containing DNA binding activities (108, 109).implies that it occurs in the absence of BCR recognition, it is This induction is relatively rapid and may provide short-termquite possible that it is mediated by weak interactions of the survival function to B cells treated with BAFF. However, long-BCR with self-molecules whose affinity is below the threshold term survival in the presence of BAFF requires kinases andto induce negative selection (at the immature stage) or to proteins of the noncanonical NF-jB pathway. Short-termtrigger self-reactivity (at the mature stage). Rajewsky and canonical NF-jB activation by BAFF may also contribute tocolleagues (102) have genetically explored the signaling BAFF-dependent survival by inducing p100 protein to serve aspathway downstream of tonic BCR signaling and found that a substrate for the noncanonical pathway. In this way, BAFFloss of the BCR on mature B cells can be compensated by treatment can initiate a positive autoregulatory loop involvingprovision of a constitutively active form of the catalytic sub- canonical and noncanonical NF-jB activation. However, theunit of PI3 kinase. In contrast, constitutively active IKK2, loop is not sufficient by itself to induce long-term BAFF-constitutively active Akt or Bcl-2 did not rescue BCR defi- dependent B-cell survival in the absence of the BCR.ciency. The mechanism by which active PI3K permits B cells Despite the critical role of the noncanonical NF-jB pathwayto respond to BAFF signaling remains unclear. It is likely in mediating BAFF-dependent survival signals, expression ofthat tonic BCR signaling is a source of constitutive NF-jB in constitutively active IKK2 (Ikk2ca), which induces canonicalmature B cells via continued degradation of IjBa (see NF-jB, in mice completely restores the B-cell deficiency inbelow). While this may be an essential function, signaling BAFF-R-deficient mice (110). This includes generation of fol-to NF-jB does not recapitulate all functions of tonic BCR licular mature and marginal zone B cells and restoration ofsignaling. splenic architecture. B cells from Ikk2ca mice have higher Published 2012. This article is a US Government work and is in the public domain in the USA260 Immunological Reviews 246/2012
  8. 8. Kaileh & Sen Æ NF-jB function in B lymphocytesnuclear levels of classical NF-jB proteins, such as RELA, but NF-jB. Additionally, high basal PI3K activity may enhancenot of p52, suggesting that generation and survival of mature responses to NF-jB-inducing stimuli during immuneB cells in these animals is entirely dependent on canonical NF- responses. The continuous requirement for PI3K activity in BjB pathway signaling. These observations suggest that synergy cells is exemplified by the absence of mature B cells in micebetween BCR and BAFF-R for B-cell survival may ultimately deficient in various components of PI3K (74, 77, 121), asfunnel through activation of classical NF-jB. Perhaps the con- well as the extreme sensitivity of mature B cells to apoptosisstitutive NF-jB DNA binding activity that is present in human upon pharmacologic inhibition of PI3K ex vivo. However, theseand mouse mature B cells reflects canonical pathway activation components have not been conditionally deleted after B-cellby both these receptors. maturation is complete to unequivocally distinguish whether they are required for differentiation and ⁄ or maintenance of mature B cells. BAFF-R signaling also induces expression of Cross-talk between BCR and BAFF-R CD21, a component of the CD19 ⁄ CD21 co-receptor complexRegulating B-cell homeostasis via two receptors provides flexi- (122, 123). This complex lowers the threshold for BCR sig-bility in modulating survival requirements during different naling thereby completing another mutually re-enforcingphases of the immune response. For this, it is imperative that loop, whereby signals through the BCR feed into the BAFF-Rthe two receptors cross-talk. This occurs at multiple levels pathway and, conversely, BAFF-R signals enhance BCR signal-between BAFF-R and the BCR. As briefly described in preced- ing. Each of these loops involves components of canonical anding section, BCR crosslinking on mature cells increases expres- noncanonical pathway NF-jB proteins.sion of the BAFF-R, thereby sensitizing cells to BAFFsignaling. In addition, NF-jB activation by of the BCR acti-vates Nfkb2 and RelB gene expression (111–113), which are NF-jB function in mature B cellssubstrates for alternate pathway activation by BAFF-R. We Beyond the essential requirement for NF-jB proteins for B-cellhave proposed that activation of p100 as a consequence of maturation and homeostasis, inducible NF-jB activation istonic BCR signaling may be an important determinant for the critical for effective immune responses. The major initiators ofdual requirement of the BCR and BAFF-R for maintenance of NF-jB signaling in B cells are the BCR, various members ofmature B cells (114). In these studies, pharmacological inhibi- the TNF receptor superfamily (in particular BAFF-R, TACI,tion of Syk tyrosine kinase activity in mature B cells ex vivo BCMA and CD40), and Toll-like receptors (in particular TLR4abrogated BCR ligand-independent upregulation of p100. Syk and 9). The constitutive nuclear NF-jB found in murine sple-inhibitor-treated cells did not maintain long-term alternate nic B cells consists primarily of REL-containing complexesNF-jB activation in response to BAFF, which correlated with (124–126); the hetero-dimeric partner is most likely p50,reduced survival of these cells. Our interpretation of these though the presence of B cells in Nfkb1-null mice indicates thatobservations is that continuous p100 protein production in p52 can substitute effectively or that REL homodimers sufficeresponse to BCR-initiated signals is essential for BAFF-depen- in the absence of p50. Similarly, REL and RELA also havedent cell survival. In follow up studies, we found that PI3K mutually compensatory functions because genetic deletion ofactivity is required for tonic, or BCR-inducible, expression of either does not impair the generation of mature B-cells. Wep100 (unpublished data, MK and RS). Thus, one way in have proposed that the predominance of REL-containing com-which PI3K activity may compensate for tonic BCR signals is plexes in B cells may be due to increased nuclear export ofby generating a pool of p100 protein that can mediate BAFF- RELA-containing complexes by IjBa because of the nuclearR-initiated survival signals. export sequence (NES) present at the C-terminus of RELA Conversely, BAFF-R-initiated signals enhance BCR signals in (127–130). REL does not contain a corresponding NES andseveral ways. For example, BAFF ⁄ BAFF-R interaction induces may therefore be better retained in the nucleus compared toPI3K and Akt activation (58, 115, 116). PI3K activity has been RELA. Indeed, B-cell differentiation and function is hamperedshown to be essential for BCR-induced NF-jB activation in B in mice whose IjBa-protein lacks an NES (131). In these micecells (117), and active forms of Akt have been shown to non-functional REL ⁄ IjBa- complexes accumulate in B-cellinduce NF-jB-dependent transcription in reporter assays nuclei, making them unavailable for IKK-dependent activation.(118–120). Continuous in vivo stimulation of B cells with This leads to reduced Nfkb2 and RelB gene expression and, as aBAFF may produce constitutively high basal PI3K activity in consequence, both canonical and noncanonical NF-jB induc-these cells that lowers the threshold of tonic BCR signaling to tion and function is impaired. These observations show thatPublished 2012. This article is a US Government work and is in the public domain in the USAImmunological Reviews 246/2012 261
  9. 9. Kaileh & Sen Æ NF-jB function in B lymphocytesshuttling of NF-jB proteins between the nucleus and significantly diminished in REL-containing complexes. Basedcytoplasm is an essential functional feature of these proteins, on additional biochemical studies, Ferch et al. (133) con-and highlight the connection between canonical and noncano- cluded that presence of MALT1 in the WT CBM complex tar-nical NF-jB signaling in the maintenance of mature B cells. gets IKK activity to REL ⁄ IjB complexes, whereas the heterodimeric CARMA1 ⁄ Bcl10 complex can target IKK only to RELA-containing complexes. An obvious implication of this NF-jB response to BCR crosslinking observation is the existence of another level of molecular rec-BCR crosslinking with anti-l chain F(ab¢)2 fragment has been ognition that distinguishes RELA- or REL complexes. Thisused as a surrogate for antigen-dependent B-cell activation. could occur, for example, by spatial segregation of RELA- orThis treatment leads to rapid NF-jB nuclear translocation via REL complexes within the cytosol such that active IKKs needthe canonical pathway. The signaling pathway that connects to be directed to different subcellular regions to activate eachthe BCR to NF-jB has been intensely studied and summarized complex. Alternatively, RELA- or REL-containing complexesin several excellent reviews (10, 132). Briefly, BCR crosslink- may need to be recruited to distinct compartments for theing activates Src tyrosine kinases leading to phosphorylation associated IjBs to be phosphorylated by IKK; in this model,of ITAM motifs in BCR-associated signaling proteins CD79a the sub-compartments that contain CB or CBM complexesand b (Iga and Igb). These phosphorylated ITAMs recruit and may be different. Finally, it is possible that REL and RELA arethereby activate the Syk kinase, which via phosphorylation of differentially associated with IjBa, b, and e. In this scenario,adapter proteins such as BLNK, Bam32, and BCAP results in ‘weakened’ NF-jB signaling via the CB complex may targetthe activation of downstream kinases cascades including one IjB better than the others, leading to induction of the RelMAPKs and PI3K. Syk also activates Btk, which is essential for proteins associated with that IjB but not the other IjBs. Somephosphorylating and activating PLCc. PLCc enzymatic activity evidence for differential REL ⁄ IjB association and its functionalresults in the generation of IP3 and diacyl glycerol; the former consequences have been previously noted in T cells (134).binds to receptors in the endoplasmic reticulum (ER) to Spatial control may also explain the essential requirementrelease calcium from ER stores and the latter is required to for PI3K in canonical NF-jB activation via the BCR. Since dele-activate protein kinase C b (PKCb). PKCb phosphorylates the tion of genes encoding PI3K catalytic or regulatory subunitsadapter CARMA1 leading to generation of a complex contain- lead to developmental defects that prevent generation ofing CARMA1, Malt1 and Bcl10 (the CBM complex) which mature B cells, the best evidence of a role for PI3K in NF-jBserves as a scaffold to bring together the IjB kinases and the activation comes from pharmacologic inhibition of PI3K dur-kinase that activates IKKs. In B cells the latter is likely to be the ing BCR crosslinking of mature wildtype cells (117). The mostTGFb-activated kinase 1 (TAK1). TAK1-mediated phosphory- likely point of intersection of PI3K with the signaling schemelation of IKK2 results in IKK2 activation, which then phospho- summarized above is Btk. Reduced NF-jB induction in B cellsrylates IjB proteins leading to their ubiquitination and from xid mice (135, 136), that express Btk protein with adegradation. As a consequence NF-jB proteins, that were point mutation in the membrane-targeting PH domain, hasbound to the IjBs are free to translocate to the nucleus and been attributed to lack of Btk activity. However, recent studiesactivate gene expression. This multi-enzyme cascade leads to show that Btk activation, as evidenced by production of phos-NF-jB activation within 30 min of BCR crosslinking in naive pho-Btk is normal in xid B cells. The problem seems to be thatmurine splenic B cells; the resulting nuclear NF-jB consists of xid Btk is unstable and present at low levels in the cells, sug-RELA- and REL-containing homo- and heterodimers. gesting that membrane recruitment by interaction with PIP3 Despite the considerable detail in which this pathway is stabilizes the protein (137). Thus, PI3K-dependent PIP3 pro-understood, some features remain unclear. One of these is the duction is a key intermediate in maintaining sufficient levelsintriguing observation that the characteristics of B cells singly of Btk. Additionally, PLCc activation by membrane-bound Btkdeficient in either CARMA1, or Bcl10 or Malt1 are not may localize active PLCc close to its substrate in the plasmaidentical with regard to NF-jB activation. In particular, Bcl10- membrane for effective function. In this manner, membranedeficient B cells do not activate IKKs (and thereby do not localization of Btk and PLCc may drive NF-jB activation ininduce NF-jB) in response to BCR crosslinking whereas Malt1- B cells. It is interesting to note that NF-jB activation in T cellsdeficient B cells activate IKKs (though not as robustly as also requires PI3K, though for different reasons. In T cells,wildtype B cells) leading to IjB degradation (133). However, non-classical PKCh is the enzyme that activates the CBMnuclear NF-jB that is induced in Malt1-deficient B cells is complex by phosphorylating CARMA1. PKCh activation is Published 2012. This article is a US Government work and is in the public domain in the USA262 Immunological Reviews 246/2012
  10. 10. Kaileh & Sen Æ NF-jB function in B lymphocytesmediated by the kinase PDK1 which, like Btk, contains a PH autoimmunity, together with elevated expression of NF-jBdomain and requires binding to PI3K-dependent PIP3 for target genes (154–156). However, the stage at which B-cellactivity (138). PI3K activity in T cells requires co-crosslinking activation is most susceptible to A20-dependent downregula-of the T-cell antigen receptor and the co-receptor CD28; in tion remains unclear. Similarly CYLD-deficiency results inB cells PI3K may be activated by BCR-associated CD19 (139, higher basal levels of NF-jB in B cells due to higher IKK2140) or activation of the adapter protein BCAP after BCR activity and increased numbers of MZ B cells (157, 158).crosslinking (141, 142). Function of Phase I NF-jB Kinetics of NF-jB activation by BCR Identifying target genes of a specific transcription factorNF-jB induced by the canonical pathway in response to BCR requires a combination of assays which must include: (i) eval-crosslinking is transient (143). This wave of NF-jB contains uation of transcript levels in the presence, or absence, of theboth RELA- and REL-containing DNA-binding proteins, transcription factor, (ii) chromatin immunoprecipitation toreaches a maximum at 1–2 h post-activation and is consider- determine transcription factor binding to important regulatoryably reduced by 6 h. Thereafter, nuclear RELA levels remain sequences of putative target genes, and (iii) evaluation of thelow despite continued presence of BCR crosslinking antibody. importance of the identified binding sites for gene transcrip-At longer time points lasting until 24 h the NF-jB response is tion. NF-jB targets in activated B cells have not been indenti-dominated by REL. We refer to these as Phases I and II of NF- fied in this comprehensive fashion; however, the identity ofjB induction and have proposed that each serves distinct func- some putative NF-jB targets affords a perspective into thetions. These observations raise two questions: (i) what is the function of phase I NF-jB. Amongst genes that were highlymechanism that restricts classical NF-jB to one cycle of activa- induced within the first 3 h were the chemokines CCL3 andtion, and (ii) what are the functions of each phase? CCL4, the chemokine receptor CCR7, the transcription factors Several possible mechanisms have been put forward for lim- IRF4 and c-Myc and the signaling proteins DUSP1 and Plk3.iting the duration of NF-jB activation in various cell types and CCL3 and 4 are interesting because they serve as chemoattrac-in response to diverse NF-jB activators (144). However, most tants for CD4+ T cells (159). By attracting CD4+ T cells, anti-of these mechanisms have not been experimentally evaluated gen exposure increases the probability of B cells to presentin BCR-activated B cells. Our working model is that the major antigens to T cells of the right specificity. Once activated suchcontributor to Phase I NF-jB downregulation is repression T cells would provide help to B cells via the CD40 ⁄ CD40Lmediated by newly-synthesized IjBa. This is the oldest model pathway. The chemokine receptor CCR7 has been implicatedof post-activation NF-jB suppression (145, 146) and is based in the movement of B cells towards the T-cell zone in theon NF-jB-dependent transcription and de novo synthesis of spleen (160), which is enriched for CCL21, the chemokineIjBa protein. The newly synthesized IjBa migrates into the ligand of CCR7. We have proposed that these Phase I NF-jBnucleus, removes DNA-bound IjBa and exports it out of the genes would maximize the possibility of B ⁄ T encounter to ini-nucleus (130, 147, 148). The cytosolic NF-jB ⁄ IjB complex tiate T-dependent B-cell immune responses. The transcriptionmay not be re-induced despite continuous BCR crosslinking factor c-Myc is essential for G1 progression of activated B cellsfor several reasons. One possibility is that re-expression of sur- (161, 162), and IRF4 is known to be required for cell growthface Ig after receptor endocytosis takes substantially longer and differentiation of B cells (163). Thus, putative NF-jB tar-(149, 150) than the duration of phase I NF-jB. Moreover, get genes induced during Phase I serve a range of functionscontinued BCR crosslinking and resulting re-endocytosis may including re-distribution of B cells, inducing cell cycle pro-prevent expression of substantial levels of the BCR to permit gression and differentiation, and altering signal transducingeffective IKK activation to induce a second cycle of canonical properties.NF-jB activation. Additional mechanisms may also reduce the One of the ways we imagine that T-dependent immuneeffectiveness of BCR signaling to NF-jB, such as inactivation responses are initiated is antigen binding to the BCR, followedof Bcl10 by degradation or phosphorylation as has been noted by endocytosis, proteolytic digestion, and expression of anti-in T cells (151, 152), or activation of de-ubiquitinating genic peptides on MHC class II molecules on the cell surface.enzymes such as A20 and CYLD (153). An important role for These MHC class II-bound peptides are recognized by T cellsA20 in B-cell physiology is evident from the observation that of the appropriate specificity, leading to activation of antigen-B-cell-specific A20 deficiency leads to hyper proliferation and specific T cells that provide B-cell help. To mimic thisPublished 2012. This article is a US Government work and is in the public domain in the USAImmunological Reviews 246/2012 263
  11. 11. Kaileh & Sen Æ NF-jB function in B lymphocytesscenario, where there is limited BCR engagement with antigen, CsA points to two well-studied transcription factors, NF-ATwe used a pulse-activation protocol to stimulate B-cells with and Mef2c, whose nuclear induction and ⁄ or transcriptionalsingle round of surface BCR signaling. Virtually the entire activity are suppressed by CsA (168–170). Though phenotyp-phase I NF-jB program was recapitulated with this form of ically Mef2c-deficient B cells resemble Rel-deficient B cells inpulse BCR-activation. Nuclear RELA and REL proteins were terms of their sensitivity to BCR-induced death and lack ofinduced with indistinguishable kinetics in pulse- versus con- BclxL mRNA induction (171), REL induction in responsetinuously activated cells, and gene expression analysis in to anti-IgM occurred normally in Mef2c-deficient B cells.pulse- or continuously activated cells showed that 70–80% of Though Rel induction has not yet been directly analyzed ininducible genes were comparably induced during the period NF-AT-deficient mice, Gerondakis and colleagues (172) haveof phase I NF-jB activation under both conditions (143). previously proposed that Rel mRNA in TCR-activated T cells isThese included 80% of putative NF-jB target genes induced induced by NF-AT. Our working hypothesis is that Rel geneover the course of the first 3 h, among which were genes induction by the BCR is also mediated by NF-AT proteins.highlighted above. Despite normal up- and down-regulationof c-Myc mRNA, however, pulse-activated B cells did notshow any evidence of G1 progression. Instead, these cells NF-jB response to CD40responded more robustly to CD40 crosslinking as evidenced T-dependent immune responses generate different classes ofby biochemical markers of G1 progression such as Cdk2 ⁄ 4 high affinity antibodies via class switch recombination (CSR)and Cyclin D2 ⁄ E expression, and phosphorylation of retino- and somatic hypermutation (SHM). These processes whichblastoma protein. Additionally anti-CD40 treatment of pulse- occur in germinal centers within the spleen and Peyer’sactivated B cells resulted in a more rapid increase of cell size patches within the gut, critically require CD40 on the B-cellcompared to naive B cells activated by CD40. Taken together, surface and induced CD40 ligand (CD40L) on CD4+ T-with activation of CCL3, CCL4 and CCR7 our working model follicular helper cells. CD40 or CD40L deficiency in mice leadsis that pulse-activation primes B cells in different ways to to defective affinity maturation of antibody responses, andreceive T-cell help. mutations in the CD40L gene in humans is one of the causes of hyper-IgM syndrome, which is associated with lack of IgG in the serum and recurrent bacterial infections. Ex vivo stimula- Characteristics of phase II NF-jB tion of naive B cells by CD40 crosslinking, or via CD40L, leadsPhase II NF-jB, which requires continuous BCR crosslinking, to cell proliferation and CSR; signaling to NF-jB is essentialis dominated by nuclear expression of REL and coincides with for these functions of novo Rel transcription and translation. Early studies showed Like other members of the TNF-receptor superfamily, CD40that long-term survival of BCR-activated cells was severely signals canonical NF-jB induction by activating IKK compleximpaired in Rel-deficient B cells due to reduced expression of via TRAF proteins (173, 174). One of the most significant dif-the anti-apoptotic genes Bcl-xL and A1 (164, 165). These ferences between CD40-induced and BCR-induced canonicalobservations are almost entirely the results of Phase II Rel NF-jB is that unlike the BCR, CD40 induces persistent NF-jBactivation. Accordingly, Bcl-xL is transiently expressed in activation (175). This NF-jB comprises of both RELA- andpulse-activated cells, while A1 expression requires continuous REL-containing DNA-binding activities. The basis for persistenttreatment with anti-IgM. Thus, a major function of Phase II NF-jB activation by CD40 has not been satisfactorily explained.NF-jB is to maintain cell viability in BCR-activated cells in Like the mechanism proposed for persistent NF-jB activationorder to permit cell division. Rel is known to be an inducible by LPS in mouse embryo fibroblasts (176, 177), one possibilitygene and has been proposed to be auto-regulated by NF-jB. is that CD40 crosslinking produces an NF-jB-inducing cyto-Our preliminary results show that phase II Rel induction kine that feeds back to reactivate NF-jB in these cells. However,occurs normally in PKCb-deficient B cells where classical NF- such a cytokine has not been identified. Alternatively, it is pos-jB activation is impaired, and in WT B cells activated in the sible that downregulatory mechanisms, such as degradation ofpresence of a pharmacologic inhibitor of IKK2. These observa- intermediate cytosolic signaling proteins, are not efficientlytions indicate that canonical NF-jB induction is not required activated after CD40 stimulation. In this scenario, post-activa-to induce phase II REL. Instead, Rel expression is sensitive to tion repression by de novo synthesized IjBa may be ineffectivecalcium chelators and is blocked by cyclosporine A (CsA) because the rate of IjBa degradation (by continued CD40 sig-treatment during BCR activation (166, 167). Sensitivity to naling) out-competes the rate of new IjBa synthesis. Published 2012. This article is a US Government work and is in the public domain in the USA264 Immunological Reviews 246/2012
  12. 12. Kaileh & Sen Æ NF-jB function in B lymphocytes Despite persistent induction of RELA, Rel-deficient B cells do LPS-induced NF-jB DNA binding and j gene transcription innot proliferate or carry out CSR in response to CD40. Thus, pre-B-cell lines was the basis of the idea that NF-jB directlyRELA apparently cannot substitute for some essential func- activated j gene transcription via the j intron enhancer. More-tion(s) of REL. The transcription factors E2F3, Myc, and IRF4 over, ‘super-induction’ of NF-jB DNA binding activity by LPShave been proposed to be Rel-responsive target genes involved in the presence of protein translational inhibitors led to thein the proliferative response; whether these genes are suffi- post-translational model (180) that is currently referred to ascient to explain the lack of proliferation of Rel) ⁄ ) B cells the canonical pathway. Super-induction of NF-jB also pre-remains to be determined. The critical role of REL in mediat- saged the idea of post-induction repression by a newly synthe-ing CD40 signals is also emphasized by the lack of organized sized inhibitor of NF-jB. LPS was used in these early studiesgerminal center formation after immunization of Rel-deficient because of its well-known property of being a B-cell mitogen.mice with T-dependent antigens as well as reduced CSR in vivo The current state of the mature B-cell response to LPS hasand in vitro (178). This effect is exacerbated in p50 ⁄ REL dou- been recently reviewed (181). LPS treatment induces REL-ble-deficient mice. It is interesting that CD40 treatment of B and RELA-containing NF-jB persistently via the canonicalcells does not induce the equivalent of Phase II REL that is pathway; the noncanonical NF-jB pathway is not activated bymediated by de novo Rel transcription and translation. This is LPS. LPS-induced proliferation is significantly reduced in Bconsistent with the observation that Rel transcription depends cells that lack Nfkb1 or Rel, though G1 progression occurs nor-on a CsA-sensitive pathway, which is not activated by CD40. mally. NFkb1- and Rel-deficient cells are also more sensitive toThus, the proliferative function of REL in CD40-treated cells is apoptosis after LPS treatment, and this effect is heightenedmediated entirely via IKK activation. considerably in Nfkb1 ⁄ Rel double-deficient cells. These obser- Stimulation via CD40 also activates the alternate NF-jB vations suggest that viability of LPS-activated B cells in syner-pathway via recruitment of TRAFs 2 and 3 leading to p100 gistically maintained by these two factors. Gerondakis anddegradation and release of p52 ⁄ RelB to activate transcription. colleagues (182) have provided a plausible mechanism for theWhereas BAFF-R-induced p52 is essential for B-cell survival cooperative effects of NFKB1 and REL on B-cell survival. Theyex vivo, CD40-induced p52 is not required for cell viability showed that NFKB1-associated Tpl1 kinase activates the ERK(175). The major function of the pathway may be to induce pathway in LPS-treated cells. Active ERK phosphorylates thechemokine genes that are required for generation and mainte- pro-apoptotic protein Bim leading to its degradation. Concur-nance of germinal centers. An important difference between rently, LPS-induced nuclear REL activates transcription of anti-CD40 and BAFF-R with regard to alternate NF-jB activation is apoptotic Bcl-XL and A1 gene expression, which neutralizethat CD40 efficiently induces expression of p100 whereas the activity of residual Bim by direct interactions. Thus, inBAFF-R does not. By constantly replenishing the store of p100 wildtype B cells reduced Bim and elevated Bcl-XL ⁄ A1 togetherrequired to generate p52, CD40 stimulation is able to main- maintain cell viability. Absence of one of the survival path-tain long-term noncanonical NF-jB activity. p100 upregula- ways in either of the single gene deficiencies results in partialtion is presumably due to sustained classical NF-jB induction sensitivity to cell death, while the absence of both survivalby CD40, but this has not been established yet. The close pathways makes Nfkb1 ⁄ Rel double-deficient cells super-sensi-working relationship between the canonical and noncanonical tive to apoptosis. Beyond maintaining cell viability, Rel is alsoNF-jB pathways in response to CD40 is exemplified by identi- essential for S-phase entry of LPS-treated B cells.fication of hypomorphic mutations in NEMO that causehyper-IgM syndrome in humans (179) that has a phenotypevery similar to deficiency of CD40L (and thereby loss of all Perspectives: conclusions and outstanding questionsCD40-dependent signaling). Jain et al. (179) showed that B Through the work of many scientists the influence of NF-jBcells from these patients induce classical NF-jB poorly, and lack now extends well beyond its originally proposed role as a jsomatic mutations and class switched Ig genes. Thus, loss of the gene activating transcription factor that is important for B-cellclassical pathway alone is sufficient to impair CD40 function. development. Yet, its essential role in generation, maintenance and function of B lymphocytes is pleasing from the notional perspective that an analysis that started with a B-cell gene is NF-jB response to LPS continuing to yield insights into B-cell biology. The most ele-Bacterial lipopolysaccharide was the first identified NF-jB- gant advances that have been made in the NF-jB field are theinducing agent (180). The close correlation between delineation of signaling pathways that connect diverse cellularPublished 2012. This article is a US Government work and is in the public domain in the USAImmunological Reviews 246/2012 265
  13. 13. Kaileh & Sen Æ NF-jB function in B lymphocytesstimuli to NF-jB. Identification of the components involved responses to strategic combinations of stimuli, which are moreand their subsequent genetic manipulation have revealed likely to represent how cells respond in vivo. Tedious though itmany biological situations that utilize NF-jB. Biochemical may be, there seems to be no way to get these insights withoutstudies of how these components function have provided tar- conditional deletions of the Rel genes themselves. Given thegets for therapeutic intervention. An area that appears ready complexity of the problem, it is reasonable to consider whatfor equally sophisticated analyses is the regulation of gene benefits (other than understanding) will accrue from suchtranscription by NF-jB. endeavors. One possibility that continues to motivate us is that At the simplest level the function of the jB site in the j uncovering subunit-specific transcriptional mechanisms mayenhancer remains mysterious. In this review, we have hypoth- make it possible to selectively alter expression of small subsetsesized possible functions for this site based on the idea that its of NF-jB target genes for therapy.high conservation between species must serve a purpose. Finally, it will be interesting to explore cross-talk betweenAdditional studies are necessary to confirm or refute these the canonical and noncanonical NF-jB pathways more deeply.conjectures. Moreover, little is known about cooperation One situation where this is pertinent is in the genera-between the jB site and other protein binding sites within the tion ⁄ maintenance of mature B cells, where both pathwaysenhancer. Obviously, this functional question cannot be have been shown to be essential. These pathways could func-addressed till we understand the function of the jB site itself. tion independently or be mutually synergistic. Our working A broader, more open-ended, question pertains to the sub- hypothesis is that they work synergistically, and the basis ofunit-specific functions of Rel family proteins. It is noteworthy synergy lies in (i) genes that are activated independently bythat many of the functional studies have been carried out with each pathway, but function cooperatively and (ii) genesconditional deletions of canonical or noncanonical signal whose transcriptional activity requires both pathways to betransducing components rather than manipulating genes activated simultaneously. A second situation where both NF-encoding Rel family members. While these studies demon- jB pathways are likely to be pertinent is in the germinal cen-strate the importance of one or the other pathway, they do not ter reaction. Two aspects of the GC reaction make it particu-readily provide insight into biological phenomena based on larly interesting: (i) CD40 activates both NF-jB pathwaysNF-jB-dependent gene expression. This applies particularly to simultaneously and persistently, which will likely be reflecteddistinguishing between the overlapping functions of REL and in the transcriptional response, and (ii) BCR signaling, andRELA. Additional complexity is introduced because REL- or accompanying classical NF-jB-dependent gene expression,RELA-dependent transcription is likely to be tissue- and signal- must be incorporated into the analysis to understand thespecific. A comprehensive understanding of NF-jB-dependent selection process that results in affinity maturation. 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