Control of lymphocyte development by nuclear factor-κB

Share Embed


Descrição do Produto

REVIEWS CONTROL OF LYMPHOCYTE DEVELOPMENT BY NUCLEAR FACTORκB Ulrich Siebenlist, Keith Brown and Estefania Claudio Abstract | The evolutionarily conserved nuclear factor-κB family of transcription factors is known to have a crucial role in rapid responses to stress and pathogens, inducing transcription of many genes that are essential for host defence. Now, studies of mice that are deficient in nuclear factor-κB-family members (or deficient in the activation of these factors) reveal that nuclear factor-κB is extensively involved in the development of T cells and B cells. And, as we review here, although these factors have several roles, their primary cell-autonomous function is to ensure lymphocyte survival at various developmental stages. This function is subverted in numerous diseases and can lead, for example, to survival of self-reactive lymphocytes or tumour cells.

RELHOMOLOGY DOMAIN

(RHD). A conserved domain of ∼300 amino acids that is found in the amino-terminal portion of nuclear factor-κB (NF-κB)-family members. It contains motifs that are responsible for dimerization, nuclear translocation and binding to NF-κB-binding motifs that are present in DNA.

Immune Activation Section, Laboratory of Immune Regulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1876, USA. Correspondence to U.S. e-mail: [email protected] doi:10.1038/nri1629 Published online 20 May 2005

The transcription factor nuclear factor-κB (NF-κB) is essential for both innate and adaptive immunity. It is crucial for the initial responses of sentinel cells to pathogens, as well as for the subsequent events that lead to T- and B-cell-mediated antigen-specific defence. The role of NF-κB in acute innate immune responses is evolutionarily conserved at least as far back as insects, in which NF-κB is responsible for the induction of nearly all responses to pathogens. Recently, NF-κB has also been recognized to be crucial for the development of several mammalian haematopoietic cell lineages and for the formation of secondary lymphoid-organ structures. In this article, we focus on the cell-autonomous roles of NF-κB in developing T and B cells, in which the main, but not exclusive, function of NF-κB is to ensure survival. The precise nature of NF-κB activity, as well as how it is activated and which genes it regulates, is context dependent, varying with the developmental stage and the initiating signal. Structure and basic regulation of NF-κB

The structure and regulation of NF-κB have recently been reviewed1–4, so we only briefly discuss these here. The mammalian NF-κB transcription-factor family (also known as the REL family) consists of p50

NATURE REVIEWS | IMMUNOLOGY

(a processing product of p105, both of which are known as NF-κB1), p52 (a processing product of p100, both of which are known as NF-κB2), REL (also known as cREL), REL-A (also known as p65) and REL-B (FIG. 1). These proteins dimerize to form functional NF-κB. All homo- and heterodimeric combinations are possible and contribute to NF-κB activity, with the exception of REL-B, which productively interacts only with p50 or p52. Family members each have a conserved RELHOMOLOGY DOMAIN (RHD), which contains three types of motif: a motif for binding specific DNA sequences; a motif for dimerization; and a motif for nuclear localization, which is known as the nuclearlocalization signal (NLS). The p50 form of NF-κB1 and the p52 form of NF-κB2 contain only an RHD, whereas REL, REL-A and REL-B also contain a TRANSACTIVATION DOMAIN. In unstimulated cells, most transactivating NF-κB dimers are inactivated by association with one of three small cytoplasmic inhibitors, inhibitor of NF-κB α (IκBα), IκBβ or IκBε (FIG. 1). These inhibitors interact, through their ankyrin domains, with NF-κB dimers, masking at least one of the NLSs. The prototypical inhibitor, IκBα, also blocks DNA binding and promotes nuclear export of bound dimers, through an amino-terminal export sequence5,6.

VOLUME 5 | JUNE 2005 | 435

© 2005 Nature Publishing Group

REVIEWS NF-κ κB family

IκB family

REL (cREL)

IκBα

REL-A (p65)

IκBβ

REL-B

IκBε

p50 (NF-κB1)

p105 (NF-κB1)

p52 (NF-κB2)

p100 (NF-κB2)

REL-homology domain Transactivation domain Ankyrin domain Degradation or processing signal site Cleavage site

BCL-3 IκBζ IκBNS

Figure 1 | The NF-κB and IκB families. The nuclear factor-κB (NF-κB) family of transcription factors in mammals consists of REL (also known as cREL), REL-A (also known as p65), REL-B, p50 (also known as NF-κB1) and p52 (also known as NF-κB2). The latter two factors are derived from longer precursors, p105 (also known as NF-κB1) and p100 (also known as NF-κB2), respectively. These factors dimerize in various combinations to form NF-κB transcription-factor complexes. The inhibitory IκB (inhibitor of NF-κB) family consists of the ‘classical’ IκBs (IκBα, IκBβ and IκBε), the NF-κB precursors (p105 and p100) and the ‘unusual’ IκBs (B-cell lymphoma 3 (BCL-3), IκBζ and IκBNS ). There are four functional domains that are conserved in these families. The first of these, the REL-homology domain (RHD), contains sequences that allow dimerization, nuclear translocation and binding to NF-κB-binding motifs in DNA. Second, the transactivation domain contains a sequence that is required for interaction with the transcriptional apparatus and for transcriptional activation. Third, the ankyrin domain contains five to seven ankyrin repeat sequences and functions by binding and inhibiting RHDs. (BCL-3 and IκBζ are exceptional because they do not bind transactivating dimers but, instead, bind p50 and p52 homodimers in nuclei; the functional consequences of this are not fully understood.) Fourth, the degradation or processing signal site contains serine residues that, when phosphorylated, trigger degradation of IκBα, IκBβ, IκBε or p105, or processing (of p100), which is mediated through ubiquitylation of residues close to the phosphorylated sites.

TRANSACTIVATION DOMAINS

Diverse structural elements that are present in transcription factors and are responsible for the activation of gene promoters. They interact with the transcriptional apparatus of the cell. PREBCR

(Pre-B-cell receptor). A receptor that is formed at the surface of pre-B cells by the pairing of rearranged immunoglobulin heavy chains with surrogate immunoglobulin light chains; it is associated with the signalling heterodimer of Igα and Igβ. Signalling by the pre-BCR possibly occurs in the absence of known ligands and is a crucial event in B-cell development.

436 | JUNE 2005

Various signals activate NF-κB by triggering degradation of IκBs, thereby freeing NF-κB dimers to enter the nucleus and bind DNA. The IκBs are first phosphorylated on two conserved serine residues by IκB kinase (IKK), a complex that is composed of a regulatory subunit, IKK-γ (also known as NEMO), and two catalytic subunits, IKK-α and IKK-β. This is followed by polyubiquitylation of IκB by the SCF (S-phase kinase-associated protein 1–Cullin-1–F-box) E3-ubiquitin-ligase complex, then destruction by proteasomes (FIG. 2). After they have been freed, NF-κB dimers translocate to the nucleus, bind their cognate DNA-binding sites (known as NF-κB-binding motifs) and induce transcription of target genes. Diverse signals activate this pathway, including the following: components of pathogens, such as lipopolysaccharide; pro-inflammatory cytokines, such as tumour-necrosis factor (TNF) and interleukin-1 (IL-1); and mitogens (FIG. 2). IKK-β- and IKK-γ-dependent activation of NF-κB through degradation of IκBs is referred to as the classical or canonical pathway of NF-κB activation. Other less well-understood pathways might liberate limited amounts of NF-κB from IκBs, including tyrosine-phosphorylation-induced dissociation of IκBs and casein-kinase-2-induced increased turnover of IκBs. After liberation, NF-κB activity can be controlled by mechanisms that affect its transactivation potency: for example, by modifications of the NF-κB subunits themselves. Activated NF-κB rapidly induces

| VOLUME 5

transcription of the gene encoding IκBα, thereby generating high levels of its own inhibitor. Newly synthesized, free IκBα enters the nucleus, removes NF-κB from DNA and exports it from the nucleus, thereby restoring the resting state. The extended IκB family also includes the precursors of the p50 and p52 forms of NF-κB1 and NF-κB2, p105 and p100, respectively (FIG. 1). In addition to the p50 and p52 sequences, these precursors contain IκB-like ankyrin domains, which inhibit NF-κB subunits that associate with them. The generation of p50 and p52 from these precursors is not completely understood, but it involves co- and/or post-translational mechanisms that require proteasomal-processing activity. Generation of p50 occurs by a constitutive co-translational mechanism, which is only partially complete and leads to approximately equal amounts of p50 and p105. Generation of p52 is largely, but not completely, a consequence of signal-induced processing of p100. Unlike the degradation of IκBα, IκBβ and IκBε, signal-induced phosphorylation and processing of p100 to p52 does not require the classical IKK-γ-dependent pathway. IKK-α and NF-κB-inducing kinase (NIK) are essential, but IKK-β and IKK-γ are not (FIG. 2). Consequently, this pathway is referred to as the non-classical, noncanonical, alternative or novel pathway of NF-κB activation. Although it is not a target of this non-classical pathway, unprocessed p105 can sometimes be targeted by the classical pathway for complete degradation, analogous to the degradation of small IκBs, thereby liberating bound NF-κB-family members. REL-B binds poorly to small IκBs, and p100 is its main inhibitor; the processing of p100 by the nonclassical pathway generates active p52–REL-B dimers. Members of the TNF superfamily induce p100 processing, including B-cell-activating factor (BAFF; also known as BLYS), CD40 ligand, lymphotoxin-α1β2 and receptor activator of NF-κB (RANK) ligand7 (also known as TRANCER) (FIG. 2). Early lymphopoiesis

It is not fully established whether NF-κB has important cell-autonomous functions in the development of early lymphocytes: that is, before the expression of the preT-cell receptor (pre-TCR) or the pre-B-cell receptor PREBCR. Nevertheless, classical-pathway-mediated activation of NF-κB might have a protective role, particularly against high levels of TNF. Haematopoietic stem cells (HSCs) from mice that are deficient in both REL-A and NF-κB1 (that is, REL-A, and p50 and p105)8, or mice that lack IKK-β9, do not generate any lymphocytes, and HSCs from mice that lack both REL-A and REL10 are mostly unable to do so. To investigate lymphopoiesis in these NF-κB-family-member-deficient mice, it was necessary to carry out adoptive transfer of their fetal liver stem cells to irradiated wild-type hosts, because mice without IKKβ or REL-A die in utero as a consequence of extensive apoptosis in the liver8,9. Haematopoietic precursors from these IKK-β- or REL-A-deficient mice fail to generate lymphocytes after transfer, because precursors are probably killed by high levels of TNF, which are generated

www.nature.com/reviews/immunol

© 2005 Nature Publishing Group

REVIEWS

Classical pathway

Non-classical pathway TNFR IL-1R

BCR

TCR

Plasma membrane

CD40, BAFFR, LT-βR or RANK

TLR

BLK

FYN

FYN LCK

LYN

TRAFs

SYK

TRAFs

ZAP70 BTK PKC-β

IKK-γ

PKC-θ

IKK-α IKK-α

NIK

IKK-β

CARMA1 BCL-10

P P

IκBα Ub Ub Ub p50 REL-A

MALT1

P P p100 REL-B Ub Ub Ub

NF-κB

IκBα fragments

NF-κB p50 REL-A

p52 REL-B

NF-κB-binding motif

NF-κB-binding motif

Nucleus

Figure 2 | Signal-transduction pathways for NF-κB activation. Nuclear factor-κB (NF-κB) is activated by signalling through many receptors. These receptors can be grouped into two classes: first, receptors that only, or mainly, activate the classical pathway of NF-κB activation; and second, receptors that activate both the classical and the non-classical pathway of NF-κB activation. The first class includes tumour-necrosisfactor receptor (TNFR), interleukin-1 receptor (IL-1R) and members of the Toll-like receptor (TLR) family. These receptors signal through various kinases and adaptors, including members of the TNFR-associated factor (TRAF) family, which are recruited to these receptors and relay signals to various downstream targets. The B-cell receptor (BCR) and T-cell receptor (TCR) activate NF-κB through a phosphorylation cascade that includes (but is not limited to) BLK (B-lymphoid kinase), FYN, LYN, SYK (spleen tyrosine kinase), BTK (Bruton’s tyrosine kinase), LCK and ZAP70 (ζ-chain-associated protein kinase of 70 kDa), which leads to activation of protein kinase C-β (PKC-β) following ligation of the BCR and PKC-θ following ligation of the TCR. (PKC-β and PKC-θ are essential for signalling through NF-κB in mature lymphocytes but not in immature lymphocytes or thymocytes). After activation of PKC, the IκB kinase (IKK) complex is activated through CARMA1 (CARD (caspase-recruitment domain)–MAGUK (membrane-associated guanylate kinase) protein 1), BCL-10 (B-cell lymphoma 10) and MALT1 (mucosa-associated lymphoid-tissue lymphoma translocation gene 1). The second class of receptors, which activate both the classical and the non-classical pathway of NF-κB activation, includes the lymphotoxin-β receptor (LT-βR), receptor activator of NF-κB (RANK), CD40 and the B-cell-activatingfactor receptor (BAFFR), although BAFFR only weakly activates the classical pathway. In the classical pathway, upstream signals induce phosphorylation of IκBα bound to cytosolic NF-κB. Phosphorylation is carried out by the IKK complex, which is composed of IKK-γ and two catalytic subunits, IKK-α and IKK-β. Phosphorylation tags IκBα for ubiquitylation and, ultimately, for proteasomal degradation, liberating NF-κB for translocation to the nucleus and activation of target genes. (Modifications of NF-κB proteins occur in the cytoplasm or nucleus to regulate activation of transcription and other functions.) The non-classical pathway is controlled through TRAFs, NF-κB-inducing kinase (NIK) and IKK-α; this regulation occurs independently of the classical IKK complex and leads to processing of the p100 form of NF-κB2, generating p52–REL-B heterodimers (and other NF-κB dimers), which migrate to the nucleus.

NATURE REVIEWS | IMMUNOLOGY

by excessive GRANULOPOIESIS. Loss of TNF or TNF receptor 1 prevents apoptosis in the liver in utero and rescues lymphopoiesis of IKK-β-deficient precursors9. Lymphopoiesis of precursors that lack both REL-A and NF-κB1 or both REL-A and REL can also be rescued if wild-type HSCs are co-transferred to recipient mice to prevent excessive TNF production8,10,11. So, NF-κB in haematopoietic cells limits the production of TNF by an unknown mechanism(s) and thereby indirectly controls lymphopoiesis. These findings further imply that high levels of TNF compromise early lymphocyte development unless NF-κB can protect these cells in a cell-autonomous manner. Additional findings support the idea that NF-κB is required for early lymphopoiesis even in the absence of abnormally high levels of TNF. The above gene-knockout mice retain compensatory IKK and NF-κB-family member activity that might help to protect cells against normal levels of TNF or other insults. Loss of the X-chromosome-encoded IKK-γ, however, completely eliminates the classical pathway of NF-κB activation. RANDOM LYONIZATION in female mice that are heterozygous for IKK-γ results in the presence of both developing wild-type cells and developing IKK-γ-deficient cells. Only wild-type (that is, IKK-γ-expressing) lymphocytes are detected in the periphery of these female mice12,13. This is also the case for women with incontinentia pigmenti, who carry one mutant IKK-γ allele14. Although it is unknown precisely when during development lympho cytes that lack IKK-γ are eliminated, the absence of IKK-γdeficient cells in the periphery indicates that it occurs early in lymphopoiesis, and this might therefore reflect a requirement for cell-autonomous, classical-pathwaymediated activation of NF-κB to protect against normal apoptotic insults. Pre-TCR and pre-BCR signals

Pre-TCR signalling and NF-κB. T-cell development in the thymus starts with CD4–CD8– double negative (DN) thymocytes, which progress to CD4+CD8+ double positive (DP) thymocytes and finally to CD4+ or CD8+ single positive (SP) thymocytes, which exit the thymus and enter the circulation15 (FIG. 3). DN CD24+ thymocytes progress through four stages: DN stage 1, at which they are CD25–CD44+; DN stage 2, CD25+CD44+; DN stage 3, CD25+CD44–; and DN stage 4, CD25–CD44–. DN cells in early stage 3 (E3) undergo TCR β-chain gene rearrangement. After rearrangement and expression, the β-chain associates with the pre-TCR-α to form the pre-TCR, thereby generating the larger DN cells in late stage 3 (L3). Signalling through the pre-TCR is required for progression to DN stage 4 and probably occurs in the absence of ligands. NF-κB seems to be important for the transition between DN stage 3 and DN stage 4 (FIG. 3). All thymocytes have some constitutive NF-κB activity, but activity is particularly high in DN stage L3 and stage 4 REFS 16,17. Interestingly, expression of a constitutively active Ikk-β transgene by RAG1 (recombination-activating gene 1)-deficient thymocytes, which

VOLUME 5 | JUNE 2005 | 437

© 2005 Nature Publishing Group

REVIEWS

Thymus

Periphery: circulation SP thymocytes CD8

CD8+ T cell CD4 Pre-TCR

TCR CD4+ T cell

• TCR and/or unknown signals transmitted through the classical pathway activate NF-κB • NF-κB is required for long-term survival of T cells from SP thymocyte stage onwards

CD25 Progenitor

DN stage 1 DN stage 2 DN stage E3 CD4–CD8– CD4–CD8– CD4–CD8– CD25–CD44+ CD25+CD44+ CD25+CD44–

DN stage L3 CD4–CD8– CD25+CD44–

DN stage 4 CD4–CD8– CD25–CD44–

• Pre-TCR signals activate NF-κB • NF-κB contributes to survival of DN cells in stage 3 and stage 4

DP thymocyte CD4+CD8+

• TCR signals activate NF-κB • NF-κB might have pro-apoptotic functions in negative selection and anti-apoptotic functions in positive selection

NF-κB is required to generate TReg cells NK1.1

NF-κB is required to generate NKT cells

Figure 3 | Role of NF-κB in T-cell development. Various stages of T-cell development in the thymus are depicted, together with some of the functions that are mediated by signal-induced activation of nuclear factor-κB (NF-κB). Dashed arrow indicates exit from thymus and entry into circulation. DN, double negative; DP, double positive; E3, early stage 3; L3, late stage 3; NKT cell, natural killer T cell; SP, single positive; TCR, T-cell receptor; TReg cell, CD4+CD25+ regulatory T cell.

GRANULOPOIESIS

The formation of granulocytes in the bone marrow. It is controlled by several cytokines, including granulocyte colony-stimulating factor (G-CSF) and granulocyte/ macrophage CSF (GM-CSF). RANDOM LYONIZATION

The random inactivation of all but one X chromosome in most cells. Iκ B SUPERREPRESSOR

A mutant form of the nuclear factor-κB (NF-κB) inhibitor IκBα. This form cannot be phosphorylated and degraded in response to signals, so it functions as a super-repressor of NF-κB, blocking its activation by upstream signals.

438 | JUNE 2005

cannot assemble the pre-TCR, allows some progression of thymocytes to DN stage 4 and to the DP stage, indicating that activation of NF-κB at DN stage 3 is important for further development. In addition, in mice that are transgenic for an Iκ B SUPERREPRESSOR, progression of thymocytes to DN stage 4 is partially blocked, and NF-κB inhibition in isolated ex vivo DN stage L3 and stage 4 cells triggers apoptosis17. So, signalling through the pre-TCR probably activates NF-κB, providing a survival signal for thymocytes in DN stage L3 and stage 4. However, whether NF-κB is essential for survival or only contributes to survival remains to be determined, because the levels of the IκB superrepressor expressed in transgenic mice only partially block NF-κB activation. Pre-BCR signalling and NF-κB. NF-κB might increase survival downstream of signalling through the preBCR in a similar manner. Prepro-B cells are the earliest recognized B-cell precursors in the bone marrow. They become pro-B cells as they begin to rearrange their immunoglobulin µ (Igµ) heavy chains. Successfully rearranged Igµ heavy chains associate with surrogate light chains (VpreB and Igλ5) to form the pre-BCR. These cells then begin to cycle and are referred to as large pre-B cells, using the Hardy nomenclature18. Large pre-B cells then become non-cycling small pre-B cells, which no longer express the pre-BCR, and these cells begin to rearrange Igκ or Igλ light chains. Rearranged Igκ or Igλ light chains combine with the rearranged Igµ heavy chains to form the BCR (also known as

| VOLUME 5

surface IgM), which is characteristic of immature B cells. These cells leave the bone marrow to mature in the spleen (FIG. 4). Recent findings indicate that NF-κB functions cellautonomously as a survival factor during the generation of small pre-B cells from pro-B cells (FIG. 4). The numbers of small pre-B cells and immature B cells are reduced when they are generated from adoptively transferred bone-marrow precursors that have been retrovirally transduced to express the IκB super-repressor19. This partial block, owing to impairment of NF-κB activity, can be overcome in cells that are transgenic for B-cell lymphoma X (BCL-X), an anti-apoptotic factor, and this provides supporting evidence of a role for NF-κB in cell survival during the generation of small pre-B cells19. Also, we have found that adoptively transferred HSCs that are deficient in NF-κB1 and NF-κB2 yield fewer small pre-B cells and immature B cells, particularly when they are in competition with wild-type cells (E.C. and U.S., unpublished observations). Signalling through the pre-BCR (possibly in a ligand-independent manner) might activate NF-κB. Evidence for this is circumstantial and is based on impaired generation of small pre-B cells in mice that lack BCR-signalling components that contribute to NF-κB activation; however, these signalling components also have other functions. Deficiency in Bruton’s tyrosine kinase (BTK) leads to fewer small pre-B cells in mice20 and to their near absence in humans21 (FIG. 2). Anti-Igβ stimulation of wild-type pre-B cells ex vivo activates NF-κB, but this does not occur in pre-B cells

www.nature.com/reviews/immunol

© 2005 Nature Publishing Group

REVIEWS

DEMETHYLATION

The removal of methyl groups from DNA. During rearrangement at immunoglobulin heavychain loci, only demethylated genomic loci are rearranged.

from mice that are deficient in B-lymphoid kinase (BLK), LYN and FYN22. These triple-deficient mice have fewer small pre-B cells and immature B cells (which have increased rates of apoptosis) and almost no mature B cells. Therefore, pre-BCR-mediated activation of NF-κB might help cells to survive during their progression to small pre-B cells. It is unknown whether this activity only contributes to survival or whether a minimum level of NF-κB activity is required (as discussed previously for the pre-TCR). NF-κB might contribute not only to survival during the large-to-small pre-B-cell transition but also to regulating rearrangement at the Igκ and Igλ loci. The Igκ locus contains a distal enhancer (3′-Eκ) and a proximal intronic enhancer (iEκ), but only the latter is known to contain an NF-κB-binding motif 23. Loss of both enhancers completely blocks Igκ lightchain rearrangement, and loss of either enhancer partially does so, indicating that these enhancers have overlapping functions that are required for rearrangement23,24. In vitro experiments indicate that iEκ, and more specifically its NF-κB-binding motif, is required for DEMETHYLATION of the Igκ locus, a step that is necessary for rearrangement of the Igκ light chain in small pre-B cells25–27. Demethylation of a transfected segment of the Igκ locus requires an intact NF-κB-binding motif and NF-κB activity, at least in one cell line that has been studied25,27. Finally, work with pre-B-cell lines transformed by Abelson murine leukaemia virus also indicates that NF-κB regulates germline transcription and rearrangement at immunoglobulin light-chain loci23. The product of the Abelson murine leukaemia virus v-abl oncogene inhibits NF-κB, as well as other factors, and prevents rearrangement at the immunoglobulin light-chain

loci. This can be overcome by stimulation of these cells with lipopolysaccharide, which induces binding of NF-κB to the NF-κB-binding motif in iEκ, and germline transcription and rearrangement at the Igκ locus. Inactivation of v-abl increases germline transcription and rearrangement at the Igκ and Igλ loci23,28,29. In both of these situations, rearrangement was dependent on NF-κB, because it was blocked by an IκB super-repressor29,30. These findings implicate NF-κB in the control of rearrangement at immunoglobulin light-chain loci, notwithstanding the fact that mice that are transgenic for the IκB superrepressor produce considerable numbers of Igκ + and Igλ+ cells. The latter finding could be accounted for by only weak inhibition of NF-κB, because B cells from these transgenic mice retain basal NF-κB activity that can be induced further31. However, a recent study provides evidence against a role for NF-κB in rearrangement at the Igκ locus, finding that mutation of the NF-κB-binding motif in iEκ does not affect this rearrangement32. Therefore, further studies are necessary to determine whether NF-κB has a role in governing Igκ-locus rearrangement. Positive and negative selection of thymocytes

CD4+CD8+ (DP) thymocytes undergo positive and negative selection (FIG. 3). Thymocytes with TCRs that bind strongly to MHC-associated self-peptides are eliminated (negative selection), as are those with TCRs that fail to bind (death by neglect). Thymocytes with TCRs that bind with lower affinity to MHCassociated self-peptides receive pro-survival signals (positive selection) and are allowed to progress to the SP stage15. NF-κB might have a role in both positive and negative selection.

Spleen

Bone marrow

PreBCR

BCR

Marginalzone B cell

IgL gene rearrangement

Pro-B cell

Late pro-B cell/ large pre-B cell

Small pre-B cell

• Pre-BCR signals activate NF-κB • NF-κB contributes to survival of small pre-B cells and initiation of Igκ and Igλ gene rearrangement

Immature B cell BCR signal might be associated with only limited activation of NF-κB, which is insufficient to protect cells and thereby leads to apoptosis and negative selection

T1 B cell

T2 B cell

• BAFFR signals transmitted through the non-classical pathway and unknown signals transmitted through the classical pathway activate NF-κB • NF-κB is required for survival of T1 and T2 B cells and for maturation

• BAFFR signals transmitted through the non-classical pathway and BCR signals transmitted through the classical pathway, as well as other signals, activate NF-κB • NF-κB is required for long-term survival of mature B cells and for generation and/or survival of marginalzone B cells

Mature B cell

Entry into circulation

Figure 4 | Role of NF-κB in B-cell development. Various stages of B2 cell (conventional B cell) development in the bone marrow and spleen are depicted, together with some of the functions that are mediated by signal-induced activation of nuclear factor-κB (NF-κB). Mature B cells complete their development in splenic follicles and can then enter the circulation and the follicles of other secondary lymphoid organs. Resident splenic marginal-zone B cells are derived from transitional B cells. The origin and developmental progression of non-conventional B cells (peritoneal B1 cells) are not well understood, but their generation and maintenance depends on B-cell receptor (BCR)-induced NF-κB activation (for details, see main text). Other B-cell stages are defined in REF. 18 and in the main text. BAFFR, B-cell-activating-factor receptor; Igκ, immunoglobulin κ light chain; Igλ, immunoglobulin λ light chain; IgL, immunoglobulin light chain; T1 B cell, B cell in transitional stage 1; T2 B cell, B cell in transitional stage 2.

NATURE REVIEWS | IMMUNOLOGY

VOLUME 5 | JUNE 2005 | 439

© 2005 Nature Publishing Group

REVIEWS

HELIXLOOPHELIX PROTEINS

(HLH proteins). Proteins that contain a particular domain (the HLH domain) that mediates dimerization between family members. This domain consists of a 40–50 amino-acid sequence that can form two amphipathic helices joined by a loop. Many transcription factors and regulatory proteins contain an HLH domain.

440 | JUNE 2005

Indirect evidence of a role for NF-κB in negative selection first emerged from studies of mice in which the IκB super-repressor is expressed specifically by T cells. Surprisingly, DP thymocytes from these mice are partially resistant to apoptosis induced by administration of CD3-specific antibodies in vivo33, as are DP thymocytes that express a dominant-negative IKK-β34. Stimulation with CD3-specific antibodies loosely mimics a strong negative-selection signal, so the data indicate that NF-κB has a pro-apoptotic function during negative selection (possibly through upregulation of expression of pro-apoptotic genes). It remains to be seen, however, whether this is a cell-autonomous function of NF-κB or whether this is mediated by NF-κB-dependent extrinsic factors. The promotion of DP-thymocyte apoptosis by NF-κB might also be deduced from transgenic mice that express inhibitors of HELIXLOOPHELIX PROTEINS in developing T cells35,36. These cells can die as a result of increased signalling through the TCR, and they are rescued in mice that are transgenic for the IκB superrepressor. A more physiological model also supports a pro-apoptotic role for NF-κB in negative selection: H-Y (male antigen)-specific TCR-transgenic male mice largely lack DP thymocytes, as a result of negative selection. However, if NF-κB is partially inhibited by an IκB super-repressor, then the number of DP thymocytes in these mice is partially rescued37. Another study indicates that negative selection is instead mediated by specific inhibition of NF-κB. In mice that express transgenic TCRs specific for positively or negatively selecting peptides, DP thymocytes that are stimulated in vivo with a negatively selecting peptide induce expression of a novel IκB-like NF-κB inhibitor — which is known as IκBNS (FIG. 1) — preceding their elimination38. This inhibitor is not induced by positively selecting peptides. It functions similar to an IκB super-repressor, because it is not subject to signal-induced degradation. When overexpressed by retroviral transduction in fetal thymic organ cultures, IκBNS causes a decrease in the number of DP and SP thymocytes, apparently by inducing apoptosis of these cells. These findings are not readily reconciled with the IκB-super-repressor-mediated protection from apoptosis (discussed earlier), although one could envisage more complex scenarios, including one in which the induction of IκBNS expression might require activation of NF-κB. NF-κB would then have both pro- and anti-apoptotic activities, depending on the timing and context. Physiological models for positive selection also implicate a role for NF-κB in this process, but during positive selection, NF-κB seems to have anti-apoptotic functions37. Certain TCR transgenes are positively selected only on specific mouse genetic backgrounds, but DP thymocytes that express such transgenes and the IκB super-repressor mostly fail to be positively selected37. Inhibition of NF-κB with the IκB super-repressor might disrupt both positive and negative selection by reducing TCR signal strength, thereby decreasing a strong negative signal that induces apoptosis to the

| VOLUME 5

level of a weaker positively selecting signal that does not, as well as decreasing a weak, positively selecting signal to the point at which it might be insufficient to allow survival. Indeed, TCR-induced phosphorylation of ZAP70 (ζ-chain-associated protein kinase of 70 kDa), a crucial intermediate in TCR signalling, is reduced when NF-κB is inhibited, although the mechanism for this is unknown37. Additional reports indicate a role for REL-B39 and IKK-α34 in the protection of DP and SP thymocytes from TCR-induced apoptosis. Further work is therefore needed to reveal the precise molecular contributions of NF-κB to positive and negative selection. Final stages of T-cell development

Recent analyses of mice that have conditional geneknockouts of Ikk-β or Ikk-γ provide insights into the roles of NF-κB in the final stages of development and in the maintenance of SP T cells (FIG. 3). In these studies, deletion of Ikk-β or Ikk-γ from T cells, or ‘knock-in’ to T cells of a gene encoding an activation-deficient IKK-β (that is, a dominant-negative IKK-β), was mediated by a Cd4-promoter-driven Cre recombinase40,41. Because complete deletion occurs slowly and across several developmental stages, and because the wildtype IKK-β or IKK-γ proteins continue to be expressed and are functional for a period after the genes encoding them have been deleted, these conditional geneknockout mice are only appropriate for analysis of late-stage T-cell development40. Conditional deletion of IKK-β has little effect on the generation and maintenance of naive peripheral T cells, although the number of peripheral CD8+ T cells is slightly reduced40. Transgenic mice in which NF-κB is partially inhibited by the IκB super-repressor have a similar phenotype42–44. However, the IKK–NF-κB pathway is, nevertheless, essential for the development of peripheral T cells, because conditional deletion of Ikk-γ or knock-in of the gene encoding activation-deficient IKK-β results in the absence of peripheral (mutant) T cells40. The defect must occur as early as the SP stage of thymocyte development (particularly for CD8+ T cells), because there are fewer of these cells in conditional Ikk-γ gene-knockout mice, apparently as a consequence of increased apoptosis40. These results indicate that some threshold of cell-autonomous IKK activity is required for the maturation and survival of SP T cells. The signal that is responsible for activation of NF-κB during this phase of T-cell development is unknown. Lack of BCL-10, a crucial signalling component of TCR-mediated NF-κB activation, does not cause loss of mature peripheral T cells, leading to the conclusion that a non-TCR signal must be involved40. Conditional loss of IKK-β results in only a small reduction in the total population of peripheral T cells (discussed earlier), probably as a result of partial compensation by IKK-α, but a closer look at individual T-cell subsets reveals that CD4+CD25+ regulatory T (TReg) cells40, natural killer T (NKT) cells 41 and memory T cells40 are greatly reduced in number in the periphery. It is not known whether these subsets

www.nature.com/reviews/immunol

© 2005 Nature Publishing Group

REVIEWS require a signal that can be delivered only through IKK-β or a level of activation that can be achieved only with IKK-β present. The dependence of these T-cell subsets on NF-κB activity is also revealed by the partial loss of TReg cells (as well as memory T cells) in mice that are deficient in both NF-κB1 and REL45, as well as by the partial loss of NKT cells in mice that are transgenic for the IκB super-repressor46. Unlike normal DP thymocytes, which are eliminated by highaffinity recognition of self-antigens, NKT cells and TReg cells are thought to be positively selected by agonist self-antigens in the thymus41. Positive selection might involve strong activation of NF-κB. In the case of TReg cells (and CD4+ memory T cells), the TCR probably provides the activation signal, because the development of these cells in the thymus requires an intact signalling pathway for TCR-mediated activation of NF-κB, including the presence of BCL-10 REFS 40,41. The development of NKT cells in the thymus is independent of these NF-κB-specific components of the TCR-signalling pathway, implying that there is either another signal, which is unknown, or a distinct TCR-induced pathway for activation of NF-κB. Negative selection of immature B cells

In the bone marrow, IgM+ (that is, BCR+) immature B cells that interact strongly with self-antigens either are eliminated by apoptosis (negative selection) or undergo further rearrangement at the immunoglobulin light-chain loci to alter their specificity. Therefore, whereas stimulation of immature B cells through the BCR increases apoptosis, it induces proliferation of mature B cells47, indicating that these cells differ in their signalling downstream of the BCR. One hypothesis is that signalling in mature B cells fully activates NF-κB and downstream signalling pathways that promote survival, whereas in immature B cells it does not47; however, this suggestion is solely based on indirect evidence. WEHI231 cells provide a possible model for negative selection of immature B cells, because they have cell-surface markers of immature B cells and because stimulation of their BCR induces apoptosis48,49. These cells show high basal NF-κB activity. After a further, transient BCR-induced increase, NF-κB activity then markedly decreases, correlating with reduced expression of cyclin D2 and MYC, as well as with cell-cycle arrest and apoptosis48,49. Rescue of NF-κB activity, by co-stimulation through CD40, was shown to block apoptosis and restore MYC expression50, indicating that apoptosis of immature B cells induced by BCR ligation is associated with inadequate NF-κB activation. Primary immature and mature B cells differ in their signalling through the BCR. For example, the BCR is recruited to lipid rafts in mature B cells but not in immature B cells. BCR signalling in immature B cells results in less efficient hydrolysis of phosphatidylinositol-4,5-bisphosphate and therefore less diacylglycerol is generated. Diacylglycerol is essential for the activation of many protein kinase C (PKC)family members, and importantly, PMA (phorbol

NATURE REVIEWS | IMMUNOLOGY

12-myristate 13-acetate)-induced activation of PKCs rescues immature B cells from BCR-induced apoptosis47. In mature B cells that are stimulated through the BCR, the diacylglycerol-dependent PKC-β isoform is required for survival, for activation of NF-κB and for induction of the anti-apoptotic factor BCL-XL51,52. PKC-β is required for the recruitment of the adaptors BCL-10 and CARMA1 (CARD (caspase-recruitment domain)–MAGUK (membrane-associated guanylate kinase) protein 1) to lipid rafts, and these are upstream components of the signalling pathway that involves the IKKs and NF-κB53 (FIG. 2). PKC-β might have a role that is analogous to the role of PKC-θ in the TCRsignalling pathway that involves IKKs and NF-κB in mature T cells54. Despite these intriguing hints, it remains to be shown whether insufficient activation of NF-κB and its target genes allows apoptosis of immature B cells stimulated through the BCR. Progression of transitional B cells

Immature B cells become transitional B cells as they leave the bone marrow and enter the spleen (FIG. 4). There, transitional B cells progress through at least two stages, T1 and T2, to become either mature follicular B cells, which can leave and enter the circulation, or non-circulating marginal-zone B cells55,56. Mature follicular B cells are also known as conventional B cells or B2 cells. Developing transitional B cells begin to move into follicles when they are in the T2 stage, the first stage at which they respond positively to stimulation through the BCR55,56. NF-κB is essential for maturation of B cells in the spleen. B cells that are deficient in both NF-κB1 and NF-κB2 REF. 57 or in both REL and REL-A10,11 are blocked in development during the transitional stage, causing an absence of mature follicular and marginalzone B cells. To circumvent the problem of early death of gene-knockout mice (and thereby allowing study of lymphocyte development), HSCs were adoptively transferred to partially irradiated RAG-deficient mice. The development of B cells that lack both NF-κB1 and NF-κB2 might be blocked slightly earlier than the development of those that lack both REL and REL-A, because the former B cells generate only T1 B cells, whereas the latter generate some T2 B cells58. This developmental defect is autonomous to B cells. When placed in culture, T1 B cells that are deficient in both NF-κB1 and NF-κB2 show an intrinsically higher rate of apoptosis59, and transitional B cells that are deficient in both REL and REL-A turn over rapidly in spleens in vivo11. Furthermore, B cells from both doubledeficient mice show decreased expression of the antiapoptotic proteins A1 (also known as BCL-2A1) and BCL-2 REFS 11,59, and exogenously expressed BCL-2 mostly rescues survival and continued maturation of B cells11. These findings clearly show that NF-κB activity is essential for survival of transitional B cells, assuring their continued developmental progression. Although transitional B cells are rescued in both types of double-deficient mouse when they are transgenic for Bcl-2, and although the continued developmental

VOLUME 5 | JUNE 2005 | 441

© 2005 Nature Publishing Group

REVIEWS progression of these cells occurs, the Bcl-2 transgene does not allow full phenotypic or functional maturation. There is not full restoration of expression of the mature B-cell surface markers CD21 and CD62, of BCRinduced proliferation or of basal (and antigen-specific) immunoglobulin production (REF. 11, and E.C. and U.S., unpublished observations). Therefore, NF-κB, in addition to assuring survival, also contributes to functional and phenotypic maturation of transitional B cells. Studies using B cells that are deficient in both NF-κB1 and NF-κB2 led to the discovery that BAFF contributes to the survival of transitional and mature B cells by stimulating processing of the p100 form of NF-κB2 (that is, the non-classical pathway of NF-κB activation). BAFF binds three receptors — TACI (transmembrane activator and CAML (calciummodulating cyclophilin ligand) interactor), BCMA (B-cell maturation antigen) and BAFFR (BAFF receptor)60 — although it is mainly the interaction of BAFF with BAFFR that is relevant for the survival of developing B cells. B-cell development in mice that are deficient in BAFF or BAFFR, or in mice that are transgenic for a TACI–immunoglobulin fusion protein (which functions as a decoy receptor for both BAFF and the BAFF-related ligand APRIL (a proliferation-inducing ligand)), is either completely blocked or mostly blocked at the transition between the T1 and T2 stages60–64. This results from a defect in cell survival, because the generation of mature follicular B cells in mice that are transgenic for a TACI–immunoglobulin fusion protein or in mice that lack BAFFR is rescued when these mice are also made transgenic for Bcl-2 REFS 62,64. In addition, it has been shown that BAFF increases the expression of BCL-2 by transitional B cells59, whereas it increases the expression of A1 and BCL-XL by mature B cells65. So, it is possible that transitional and mature B cells differ in their response to BAFF. For mature B cells, the survival role of BAFF might also involve a block in the (spontaneous) nuclear localization of PKC-δ, which has pro-apoptotic functions in the nucleus66. In some experimental settings, BAFF-mediated survival entirely depends on the non-classical pathway of NF-κB activation. BAFF promotes survival of transitional B cells ex vivo, but not if these transitional B cells lack NF-κB2 (that is, both p52 and p100) or carry the alymphoplasia mutation (Nikaly/Nikaly) in NIK, which is an integral component of the non-classical pathway59. Furthermore, BAFF potently induces processing of p100 in immature, transitional and mature B cells, leading to nuclear translocation of p52–REL-B complexes. Nevertheless, the non-classical pathway might not be the only means by which BAFF contributes to the survival of developing B cells in vivo. The absence of BAFF completely blocks B-cell development, and the absence of BAFFR mostly does so; however, loss of the non-classical pathway by functional inactivation of IKK-α or NIK (as occurs in Nikaly/Nikaly mice) leads to only a partial reduction in the number of mature B cells59,67–69. Lack of NF-κB2 causes the smallest reduction in the number of mature B cells, probably because this eliminates not only p52 but also p100, the main inhibitor of REL-B.

442 | JUNE 2005

| VOLUME 5

In addition to the non-classical pathway, the classical IKK-β- and IKK-γ-dependent pathway also contributes to the survival of transitional B cells in vivo. Conditional Ikk-γ gene-knockout in B cells or conditional knock-in of an Ikk-β mutant in B cells reduces the number of transitional B cells in the spleen (and the number of mature B cells)70. Loss of both NF-κB1 and NF-κB2 might completely block progression at the transitional stage (discussed earlier), because both the classical and the non-classical pathways of NF-κB activation are simultaneously compromised. In the absence of only the non-classical pathway, an NF-κB1 (p50 and p105)dependent mechanism must be able to compensate in vivo, although the signal for this is unknown. There is no evidence that the BCR activates NF-κB at the T1 stage of development, although this pathway might contribute to a later step in B-cell maturation. BTK is a crucial component of BCR-induced NF-κB activation in mature B cells (FIG. 2), and BTK-deficient mice are partially blocked in the transition from T2 B cells to mature B cells71, indicating that BCR-induced NF-κB activation is important at this stage. Maintenance of mature B cells

Although the loss of only certain combinations of genes encoding NF-κB subunits completely blocks the generation of mature follicular B cells, the generation and/or maintenance of marginal-zone B cells is completely (through loss of NF-κB1, NF-κB2 or REL-B) or partially (through loss of REL or REL-A) blocked in mice that are deficient in a single factor59,72–73. Also, the mature follicular B cells that are generated in mice that are deficient in a single factor have particular survival defects, highlighting that NF-κB has crucial functions for survival in general and that individual NF-κB subunits have survival functions in specific situations. For example, when compared with wildtype cells, deficiency in an NF-κB subunit results in the following phenotypes: NF-κB1-deficient mature B cells turn over more rapidly in vivo and have an increased intrinsic apoptotic rate ex vivo74; REL-deficient mature B cells are impaired in BCR-stimulated proliferation and survival ex vivo74; REL-A-deficient B cells are particularly sensitive to TNF-induced apoptosis75; and NF-κB2-deficient transitional and mature B cells have impaired survival ex vivo59. The longevity of mature B cells probably depends on several input signals, including signals from the BCR and BAFFR. The survival signals that are elicited by ligation of these receptors are at least partly mediated by NF-κB, and they involve both the classical and the non-classical pathways of NF-κB activation. The BCR is essential for the maintenance of mature B cells, given the complete loss of all mature B cells in which the genes encoding the BCR are conditionally knocked out76. NF-κB is a relevant target for signals from the BCR, because mice that are deficient in adaptors required for BCR-mediated activation of NF-κB — that is, CARMA1, BCL-10 and MALT1 (mucosaassociated lymphoid-tissue lymphoma-translocation gene 1) — have a reduced number of mature B2 cells

www.nature.com/reviews/immunol

© 2005 Nature Publishing Group

REVIEWS and few marginal-zone B cells or peritoneal B1 cells54,77. Conditional gene-knockout of Ikk-β 78 or Ikk-γ 70 also clearly implicates the classical pathway as essential for maintenance of peripheral mature B cells. Most of the few surviving mature B cells and nearly all marginalzone B cells in these mice still expressed IKK-β or IKK-γ, indicating that loss of these proteins is incompatible with an extended lifespan of mature B cells. However, when the influx of newly generated B cells into the periphery of these mice is blocked with IL-7-specific antibodies, then even these remaining cells disappear rapidly as their Ikk-β or Ikk-γ alleles are finally deleted70,78. A probable role for NF-κB in maintaining the peripheral B-cell pool also emerges from earlier studies of transgenic mice that have B cells expressing the IκB super-repressor31 and from studies of mice that are deficient in both REL and NF-κB1 (p50 and p105), as these mice have moderately reduced numbers of recirculating B cells79. Finally, the nonclassical pathway of activation of NF-κB contributes to the normal longevity of B cells. RADIATION CHIMERAS of mice that are deficient in IKK-α or mice that have undergone knock-in of a mutant Ikk-α 67,68 are impaired in the processing of NF-κB2 from p100 to p52 and have fewer mature, recirculating B cells, and these remaining B cells turn over more rapidly in vivo, express less of the anti-apoptotic factor A1 and have a reduced capacity to survive ex vivo. Because BAFF promotes survival of mature B cells in the periphery, BAFF-induced, IKK-αmediated processing of p100 therefore contributes to normal life expectancy and, consequently, to maintenance of mature B cells. Interestingly, although the conventional B cells that we have discussed here (that is, B2 cells) depend on BAFF–BAFFR signalling, peritoneal B1 cells do not60. Generation and/or maintenance of B1 cells does, however, depend on BCR-mediated activation of NF-κB, because loss of CARMA1, BCL-10 or MALT1 markedly reduces their numbers54,77, with some reduction also noted in mice that are deficient in both REL and NF-κB1 REF. 79. Perspectives

RADIATION CHIMERAS

Animals that contain cell populations of different genotypes as a result of the transfer of haematopoietic stem cells from fetal liver or bone marrow to a recipient in which haematopoietic cell populations (and other actively dividing cell populations) have been fully or partially destroyed by lethal or sub-lethal ionizing radiation.

NF-κB is important for the survival of developing lymphocytes. During some developmental transitions, specific NF-κB activities are required for survival, whereas other transitions seem merely to be helped by NF-κB activity. This is revealed using gene-knockout mouse models in which lymphocytes lack a specific NF-κB subunit or signalling component. However, if lymphocytes that are partially impaired in NF-κB activity need to compete with wild-type counterparts, then the loss of a ‘helping’ contribution can become crucial. This ensures that only those lymphocytes that have optimal NF-κB activity will develop and be maintained. Furthermore, only lymphocytes with intact NF-κB systems will become part of the peripheral pool and face pathogenic threats. NF-κB can influence lymphocyte development through its functions in many cell types, but in this article, we have emphasized self-autonomous functions. The pervasive role of NF-κB is to help

NATURE REVIEWS | IMMUNOLOGY

developing lymphocytes survive. At times, NF-κB is needed to counter cell-death signals that are induced by extrinsic factors (such as TNF), whereas at other times, survival signals (such as BAFF) activate NF-κB and allow cell death by default to be avoided. The latter mechanism thereby rescues only those cells that progress and migrate to the proper location for receiving the required input signal. NF-κB induces expression of several survival genes, including (but not solely) anti-apoptotic BCL-2-family members. Which survival pathway(s) is relevant might depend on the context: that is, the cell type, developmental stage or initiation signal. In early lymphocyte development, a minimum of NF-κB activity might be sufficient, and subunits could readily substitute for each other; by contrast, higher levels and/or more selective contributions of NF-κB activity are required during the later stages of development and in mature cells. Different signals activate distinct NF-κB dimers and thereby induce distinct targets: for example, BAFFR ligation activates the non-classical pathway and p52–REL-B dimers, whereas antigen-receptor ligation activates the classical pathway and mainly p50–REL and p50–REL-A dimers. In addition to survival, NF-κB also contributes to expression of phenotypic markers and probably to proliferation, particularly during late B-cell development. It is in B cells that NF-κB might have its greatest impact and in which the full spectrum of subunits and activities of this transcription-factor family operates. NF-κB is well known for its transcriptional induction of host-defence mediators during acute pathogenic threats. Equally important is the ability of NF-κB to promote cell survival, countering extrinsic and intrinsic death threats in mature cells, as well as in developing lymphocytes. These normal functions of NF-κB can be subverted in a variety of diseases. Autoimmune diseases might be initiated in malfunctioning lymphocytes when apoptotic pathways that are activated by self-antigens are blocked by abnormal activation of NF-κB, enabling the survival of self-reactive cells80–83. Inflammation has been linked to many cancers84,85, and abnormal, inflammation-driven activation of NF-κB (not only in tumour cells but also in the surrounding cells) contributes to the survival of developing or progressing tumours84–89. The broad involvement of NF-κB in cancer and inflammatory and autoimmune diseases has made NF-κB an important pharmaceutical target. Given its many crucial roles in maintaining health, including roles in acute host defence and lymphocyte development, broadly acting NF-κB inhibitors are likely to have side-effects, particularly if they are used for a long time. Such broadly acting inhibitors might still be useful if they can be administered in doses that interfere with disease progression while sparing normal processes. More promising are inhibitors that target a specific subunit of NF-κB or the pathway(s) that leads to its activation in a particular disease. To discover such targets and inhibitors, we need to improve our understanding of the roles of NF-κB and its pathways of activation in healthy and diseased cells.

VOLUME 5 | JUNE 2005 | 443

© 2005 Nature Publishing Group

REVIEWS

1. 2. 3. 4.

5.

6.

7.

8.

9.

10.

11.

12. 13.

14.

15. 16.

17.

18. 19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Li, Q. & Verma, I. M. NF-κB regulation in the immune system. Nature Rev. Immunol. 2, 725–734 (2002). Ghosh, S. & Karin, M. Missing pieces in the NF-κB puzzle. Cell 109, S81–S96 (2002). Hayden, M. S. & Ghosh, S. Signaling to NF-κB. Genes Dev. 18, 2195–2224 (2004). Li, Z.-W., Rickert, R. C. & Karin, M. Genetic dissection of antigen receptor induced-NF-κB activation. Mol. Immunol. 41, 701–714 (2004). Tam, W. F., Lee, L. H., Davis, L. & Sen, R. Cytoplasmic sequestration of Rel proteins by IκBα requires CRM1dependent nuclear export. Mol. Cell. Biol. 20, 2269–2284 (2000). Lee, S.-H. & Hannink, M. The N-terminal nuclear export sequence of IκBα is required for RanGTP-dependent binding to CRM1. J. Biol. Chem. 276, 23599–23606 (2001). Bonizzi, G. & Karin, M. The two NF-κB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25, 280–288 (2004). Horwitz, B. H., Scott, M. L., Cherry, S. R., Bronson, R. T. & Baltimore, D. Failure of lymphopoiesis after adoptive transfer of NF-κB-deficient fetal liver cells. Immunity 6, 765–772 (1997). Senftleben, U., Li, Z. W., Baud, V. & Karin, M. IKKβ is essential for protecting T cells from TNFα-induced apoptosis. Immunity 14, 217–230 (2001). Grossmann, M. et al. The combined absence of the transcription factors Rel and RelA leads to multiple hemopoietic cell defects. Proc. Natl Acad. Sci. USA 96, 11848–11853 (1999). Grossmann, M. et al. The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression. EMBO J. 19, 6351–6360 (2000). Schmidt-Supprian, M. et al. NEMO/IKKγ-deficient mice model incontinentia pigmenti. Mol. Cell 5, 981–989 (2000). Makris, C. et al. Female mice heterozygous for IKKγ/ NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol. Cell 5, 969–979 (2000). Smahi, A. et al. The NF-κB signalling pathway in human diseases: from incontinentia pigmenti to ectodermal dysplasias and immune-deficiency syndromes. Hum. Mol. Genet. 11, 2371–2375 (2002). Germain, R. N. T-cell development and the CD4–CD8 lineage decision. Nature Rev. Immunol. 2, 309–322 (2002). Sen, J. et al. Expression and induction of nuclear factor-κB-related proteins in thymocytes. J. Immunol. 154, 3213–3221 (1995). Voll, R. E. et al. NF-κB activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development. Immunity 13, 677–689 (2000). Pre-TCR signals that activate NF-κB provide a survival signal for thymocytes. Hardy, R. R. & Hayakawa, K. B cell development pathways. Annu. Rev. Immunol. 19, 595–562 (2001). Feng, B., Cheng, S., Pear, W. S. & Liou, H. C. NF-κB inhibitor blocks B cell development at two checkpoints. Med. Immunol. 3, 1 (2004). Middendorp, S., Dingjan, G. M. & Hendriks, R. W. Impaired precursor B cell differentiation in Bruton’s tyrosine kinasedeficient mice. J. Immunol. 168, 2695–2703 (2002). Conley, M. E., Rohrer, J., Rapalus, L., Boylin, E. C. & Minegishi, Y. Defects in early B-cell development: comparing the consequences of abnormalities in pre-BCR signaling in the human and the mouse. Immunol. Rev. 178, 75–90 (2000). Saijo, K. et al. Essential role of Src-family protein tyrosine kinases in NF-κB activation during B cell development. Nature Immunol. 4, 274–279 (2003). Schlissel, M. S. Regulation of activation and recombination of murine Igκ locus. Immunol. Rev. 200, 215–223 (2004). Xu, Y., Davidson, L., Alt, F. W. & Baltimore, D. Deletion of the Igκ light chain intronic enhancer/matrix attachment region impairs but does not abolish VκJκ rearrangement. Immunity 4, 377–385 (1996). Kirillov, A. et al. A role of nuclear NF-κB in B-cell-specific demethylation of the Igκ locus. Nature Genet. 4, 435–441 (1996). Mostoslavsky, R. et al. Demethylation and the establishment of κ allelic exclusion. Cold Spring Harb. Symp. Quant. Biol. 64, 197–206 (1999). Goldmit, M. & Bergman, Y. Monoallelic gene expression: a repertoire of recurrent themes. Immunol. Rev. 200, 197–214 (2004). Muljo, S. A. & Schlissel, M. S. A small molecule Abl kinase inhibitor induces differentiation of Abelson virus-transformed pre-B cell lines. Nature Immunol. 4, 31–37 (2003).

444 | JUNE 2005

29. Bendall, H. H., Sikes, M. L. & Oltz, E. M. Transcription factor NF-κB regulates Igλ light chain gene rearrangement. J. Immunol. 167, 264–269 (2001). 30. Scherer, D. C. et al. Corepression of RelA and c-Rel inhibits immunoglobulin κ gene transcription and rearrangement in precursor B lymphocytes. Immunity 5, 563–574 (1996). 31. Bendall, H. H., Sikes, M. L., Ballard, D. W. & Oltz, E. M. An intact NF-κB signaling pathway is required for maintenance of mature B cell subsets. Mol. Immunol. 36, 187–195 (1999). 32. Inlay, M. A., Tian, H., Lin, T. & Xu, Y. Important roles for E protein binding sites within the immunoglobulin κ chain intronic enhancer in activating VκJκ rearrangement. J. Exp. Med. 200, 1205–1211 (2004). 33. Hettmann, T., DiDonato, J., Karin, M. & Leiden, J. M. An essential role for nuclear factor κB in promoting double positive thymocyte apoptosis. J. Exp. Med. 189, 145–158 (1999). 34. Ren, H., Schmalstieg, A., van Oers, N. S. & Gaynor, R. B. I-κB kinases α and β have distinct roles in regulating murine T cell function. J. Immunol. 168, 3721–3731 (2002). 35. Kim, D., Peng, X. C. & Sun, X. H. Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice. Mol. Cell. Biol. 19, 8240–8253 (1999). 36. Kim, D. et al. Helix–loop–helix proteins regulate pre-TCR and TCR signaling through modulation of Rel/NF-κB activities. Immunity 16, 9–21 (2002). 37. Mora, A. L., Stanley, S., Armistead, W., Chan, A. C. & Boothby, M. Inefficient ZAP-70 phosphorylation and decreased thymic selection in vivo result from inhibition of NF-κB/Rel. J. Immunol. 167, 5628–5635 (2001). IκB super-repressor-mediated inhibition of NF-κB interferes with both negative and positive selection of thymocytes in vivo. 38. Fiorini, E. et al. Peptide-induced negative selection of thymocytes activates transcription of an NF-κB inhibitor. Mol. Cell 9, 637–648 (2002). 39. Guerin, S. et al. RelB reduces thymocyte apoptosis and regulates terminal thymocyte maturation. Eur. J. Immunol. 32, 1–9 (2002). 40. Schmidt-Supprian, M. et al. Mature T cells depend on signaling through the IKK complex. Immunity 19, 377–389 (2003). IKK and the classical pathway of NF-κB activation are essential for the generation and survival of mature T cells. The development of regulatory and memory T cells specifically depends on IKK-β. 41. Schmidt-Supprian, M. et al. Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-κB activation. Proc. Natl Acad. Sci. USA 101, 4566–4571 (2004). 42. Esslinger, C. W., Wilson, A., Sordat, B., Beermann, F. & Jongeneel, C. V. Abnormal T lymphocyte development induced by targeted overexpression of IκBα. J. Immunol. 158, 5075–5078 (1997). 43. Boothby, M. R., Mora, A. L., Scherer, D. C., Brockman, J. A. & Ballard, D. W. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-κB. J. Exp. Med. 185, 1897–1907 (1997). 44. Attar, R. M., Macdonald-Bravo, H., Raventos-Suarez, C., Durham, S. K. & Bravo, R. Expression of constitutively active IκBβ in T cells of transgenic mice: persistent NF-κB activity is required for T-cell immune responses. Mol. Cell. Biol. 18, 477–487 (1998). 45. Zheng, Y., Vig, M., Lyons, J., Van Parijs, L. & Beg, A. A. Combined deficiency of p50 and cRel in CD4+ T cells reveals an essential requirement for nuclear factor κB in regulating mature T cell survival and in vivo function. J. Exp. Med. 197, 861–874 (2003). 46. Sivakumar, V., Hammond, K. J., Howells, N., Pfeffer, K. & Weih, F. Differential requirement for Rel/nuclear factor κB family members in natural killer T cell development. J. Exp. Med. 197, 1613–1621 (2003). 47. King, L. B. & Monroe, J. G. Immunobiology of the immature B cell: plasticity in the B-cell antigen receptorinduced response fine tunes negative selection. Immunol. Rev. 176, 86–104 (2000). 48. Wu, M. et al. Inhibition of NF-κB/Rel induces apoptosis of murine B cells. EMBO J. 15, 4682–4690 (1996). 49. Banerji, L. et al. BCR signals target p27 (Kip1) and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells. Oncogene 20, 7352–7367 (2001). 50. Donjerkovic, D. & Scott, D. W. Activation-induced death in B lymphocytes. Cell Res. 10, 179–192 (2000). 51. Saijo, K. et al. Protein kinase C β controls nuclear factor κB activation in B cells through selective regulation of the IκB kinase α. J. Exp. Med. 195, 1647–1652 (2002).

| VOLUME 5

52. Guo, B., Su, T. T. & Rawlings, D. J. Protein kinase C family functions in B-cell activation. Curr. Opin. Immunol. 16, 367–373 (2004). 53. Su, T. T. et al. PKC-β controls IκB kinase lipid raft recruitment and activation in response to BCR signaling. Nature Immunol. 3, 780–786 (2002). 54. Thome, M. CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nature Rev. Immunol. 4, 348–359 (2004). 55. Cancro, M. P. Peripheral B-cell maturation: the intersection of selection and homeostasis. Immunol. Rev. 197, 89–101 (2004). 56. Rathmell, J. C. B cell homeostasis: digital survival or analog growth? Immunol. Rev. 197, 116–128 (2002). 57. Franzoso, G. et al. Requirement for NF-κB in osteoclast and B-cell development. Genes Dev. 11, 3482–3496 (1997). 58. Gerondakis, S. & Strasser, A. The role of Rel/NF-κB transcription in B lymphocyte survival. Semin. Immunol. 15, 159–166 (2003). 59. Claudio, E., Brown, K., Park, S., Wang, H. & Siebenlist, U. BAFF-induced NEMO-independent processing of NF-κB2 in maturing B cells. Nature Immunol. 10, 958–965 (2002). BAFFR activates the non-classical pathway of NF-κB activation, which is important for the survival and maturation of transitional B cells. 60. Mackay, F. & Browning, J. L. BAFF: a fundamental survival factor for B cells. Nature Rev. Immunol. 7, 465–475 (2002). 61. Gross, J. A. et al. TACI–Ig neutralizes molecules critical for B cell development and autoimmune disease: impaired B cell maturation in mice lacking BLyS. Immunity 15, 289–302 (2001). 62. Sasaki, Y., Casola, S., Kutok, J. L., Rajewsky, K. & Schmidt-Supprian, M. TNF family member B cell-activating factor (BAFF) receptor-dependent roles for BAFF in B cell physiology. J. Immunol. 173, 2245–2252 (2004). 63. Shulga-Morskaya, S. et al. B cell activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation. J. Immunol. 173, 348–359 (2004). 64. Tardivel, A. et al. The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF. Eur. J. Immunol. 34, 509–518 (2004). 65. Hsu, B. L., Harless, S. M., Lindsley, R. C., Hilbert, D. M. & Cancro, M. P. BlyS enables survival of transitional and mature B cells through distinct mediators. J. Immunol. 168, 5993–5996 (2002). 66. Mecklenbrauler, I., Kalled, S. L., Leitges, M., Mackay, F. & Tarakhovsky, A. Regulation of B-cell survival by BAFFdependent PKCδ-mediated nuclear signaling. Nature 431, 456–461 (2004). Spontaneous death of resting B cells is controlled by nuclear localization of PKC-δ. Treatment of cells with BAFF prevents the nuclear accumulation of PKC-δ. 67. Senftleben, U. et al. Activation by IKKα of a second, evolutionarily conserved, NF-κB signaling pathway. Science 293, 1495–1499 (2001). IKK-α is a component of the non-classical pathway of NF-κB activation. This pathway leads to the processing of p100 into p52 rather than IκB degradation. 68. Kaisho, T. et al. IκB kinase α is essential for mature B cell development and function. J. Exp. Med. 193, 417–426 (2001). IKK-α-chimeric mice have disrupted B-cell zones in the spleen, and IKK-α-deficient B cells have impaired survival and mitogenic responses in vitro. 69. Yamada, T. et al. Abnormal immune function of hemopoietic cells from alymphoplasia (aly) mice, a natural strain with mutant NF-κB-inducing kinase. J. Immunol. 165, 804–812 (2000). 70. Pasparakis, M., Schmidt-Supprian, M. & Rajewsky, K. IκB kinase signaling is essential for maintenance of mature B cells. J. Exp. Med. 196, 743–752 (2002). Conditional knockout of Ikk-β or Ikk-γ from B cells reveals the importance of the classical pathway of NF-κB activation for maintenance of peripheral B cells, beginning with defects in transitional B cells. 71. Khan, W. N. Regulation of B lymphocyte development and activation by Bruton’s tyrosine kinase. Immunol. Res. 23, 147–156 (2001). 72. Cariappa, A., Liou, H. C., Horwitz, B. H. & Pillai, S. Nuclear factor κB is required for the development of marginal zone B lymphocytes. J. Exp. Med. 192, 1175–1182 (2000). 73. Weih, D. S., Yilmaz, Z. B. & Weih, F. Essential role of RelB in germinal center and marginal zone formation and proper expression of homing chemokines. J. Immunol. 167, 1909–1919 (2001).

www.nature.com/reviews/immunol

© 2005 Nature Publishing Group

REVIEWS

74. Grumont, R. J. et al. B lymphocytes differentially use the Rel and nuclear factor κB1 (NF-κB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells. J. Exp. Med. 187, 663–674 (1998). 75. Prendes, M., Zheng, Y. & Beg, A. A. Regulation of developing B cell survival by RelA-containing NF-κB complexes. J. Immunol. 171, 3963–3969 (2003). 76. Kraus, M., Alimzhanov, M. B., Rajewsky, N. & Rajewsky, K. Survival of resting mature B lymphocytes depends on BCR signaling via the Igα/β heterodimer. Cell 117, 787–800 (2004). 77. Xue, L. et al. Defective development and function of Bcl10-deficient follicular, marginal zone and B1 B cells. Nature Immunol. 4, 857–864 (2003). BCL-10-deficient mice show impaired progression of transitional B cells to mature B cells, as well as a decrease in the number of marginal-zone B cells and B1 cells. BCL-10-deficient follicular and marginal-zone B cells also fail to proliferate. So, BCL-10 is essential for the development of all mature B-cell subsets. 78. Li, Z.-W., Omori, S. A., Labuda, T., Karin, M. & Rickert, R. C. IKKβ is required for peripheral B cell survival and proliferation. J. Immunol. 170, 4630–4637 (2003). Loss of IKK-β from B cells severely reduces the number of cells in all peripheral B-cell subsets. IKK-β β-deficient B cells have impaired responses, indicating that there is a role for this protein in the activation and maintenance of B cells.

79. Pohl, T. et al. The combined absence of NF-κB1 and c-Rel reveals that overlapping roles for these transcription factors in the B cell lineage are restricted to the activation and function of mature cells. Proc. Natl Acad. Sci. USA 99, 4514–4519 (2002). 80. Kayagaki, N. et al. BAFF/BLyS receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface loop and promotes processing of NF-κB2. Immunity 17, 515–524 (2002). 81. Mackay, F. et al. Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J. Exp. Med. 190, 1697–1710 (1999). 82. Khare, S. D. et al. Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc. Natl Acad. Sci. USA 97, 3370–3375 (2000). 83. Gross, J. A. et al. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404, 995–999 (2000). 84. Greten, F. R. et al. IKKβ links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 118, 285–296 (2004). 85. Pikarsky, E. et al. NF-κB functions as a tumour promoter in inflammation-associated cancer. Nature 431, 461–466 (2004). 86. Davis, R. E., Brown, K. D., Siebenlist, U. & Staudt, L. M. Constitutive nuclear factor κB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells J. Exp. Med. 194, 1861–1874 (2001).

NATURE REVIEWS | IMMUNOLOGY

87. Lam, L. T. et al. Small molecule inhibitors of IκB kinase are selectively toxic for subgroups of diffuse large B-cell lymphoma defined by gene expression profiling. Clin. Cancer Res. 11, 28–40 (2005). 88. Krappmann, D. et al. Molecular mechanisms of constitutive NF-κB/Rel activation in Hodgkin/ReedSternberg cells. Oncogene 18, 943–953 (1999). 89. Hinz, M. et al. Nuclear factor κB-dependent gene expression profiling of Hodgkin’s disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity. J. Exp. Med. 196, 605–617 (2002).

Acknowledgements We thank all members, past and present, of the Siebenlist laboratory for their contributions.

Competing interests statement The authors declare no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene IκBα | IκBβ | IκBε | NF-κB1 | NF-κB2 | REL | REL-A | REL-B Access to this interactive links box is free online.

VOLUME 5 | JUNE 2005 | 445

© 2005 Nature Publishing Group

Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.