5 n fkapa b

Published 2012. This article is a US Government work and is in the public domain in the USA254 Immunological Reviews 246/2012

Mary Kaileh

Ranjan SenNF-jB function in B lymphocytes

Authors’ address

Mary Kaileh1, Ranjan Sen1

1Gene Regulation Section, Laboratory of Molecular Biology

and Immunology, National Institute on Aging, National Insti-

tutes of Health, Baltimore, MD, USA.

Correspondence to:

Ranjan Sen

Gene Regulation Section

Laboratory of Molecular Biology and Immunology

National Institute on Aging

National Institutes of Health

251 Bayview Boulevard

Baltimore, MD 21224, USA

Tel.: +410 558 8630

Fax: +410 558 8386

e-mail: [email protected]

Acknowledgements

The authors are supported entirely by the Intramural

Research Program of the NIH, National Institute on Aging

(Baltimore, MD). The authors have no conflicts of interest to

declare.

This article is part of a series of

reviews covering NF-jB appearing in

Volume 246 of Immunological Reviews.

Video podcast available

Go to

www.immunologicalreviews.com to

watch interview with Guest Editor

Sankar Ghosh

Immunological Reviews 2012

Vol. 246: 254–271

Printed in Singapore. All rights reserved

Published 2012. This article is a US Government workand is in the public domain in the USA

Immunological Reviews

0105-2896

Summary: NF-jB proteins were identified in the search for mecha-nisms that regulate B-lymphocyte-specific transcription of immunoglob-ulin j light chain genes. Twenty-five years later, though the function ofthe jB site in the enhancer remains enigmatic, NF-jB proteins have beenshown to have important roles in B-cell development, maintenance, andfunction. 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-phocytes for the general reader is followed by three sections that examinethe role of NF-jB family of proteins in B-cell development, mature B-cellsurvival and B-cell function. We endeavor throughout to suggest mecha-nisms and implications of the wide-ranging observations that have beenmade and conclude by highlighting the need to understand NF-jB-medi-ated gene expression in more depth.

Keywords: NF-jB, B lymphocytes, BCR, function, development

Overview of B-cell biology

The adaptive immune system is characterized by the ability to

respond to pathogens that have never been encountered

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

of the immune system and the specially designed antigen

receptors that are expressed by these cells. Unlike cells of the

innate system that recognize limited numbers of pathogen-

associated molecular patterns, each B and T lymphocyte

expresses a unique antigen receptor. Thereby, lymphocytes

provide millions of potential antigen-recognition specificities,

and generation of a diverse repertoire of lymphocytes is a key

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-

specific cells must be preserved to provide memory. Thus, the

history of lymphocytes consists of development, survival for a

finite time period, selection by antigens for expansion and

function, and long-term survival of memory.

For B cells, development starts with hematopoietic stem

cells present in the fetal liver or the adult bone marrow. Fetal

B lymphopoiesis occurs in the fetal liver and provides the neo-

nate with B cells. The functionality of these cells remains an

area of much study, however it is very likely that they provide

a degree of protection soon after birth (1). For much of the

lifetime of vertebrates, B lymphopoiesis occurs in the adult

bone marrow. A difference between B lymphocytes produced

in the fetal liver and the bone marrow is the specificity of their

antigen receptor repertoires. Generation of antigen receptor

diversity occurs at the distinct steps of B-cell development via

stage-specific rearrangement of immunoglobulin (Ig) heavy

(H) and light (L) chains genes (2–4). IgH gene assembly takes

place first in pro-B cells (Fig. 1). Because V(D)J recombination

is error-prone, not all cells that initiate IgH gene rearrange-

ments successfully produce IgH protein. IgH+ and IgH) cells

are distinguished on the basis of signals transduced via a pre-

B-cell receptor (pre-BCR) composed of IgH chains and light-

chain-like molecules k5 and V-pre-B. pre-BCR+ cells undergo

several rounds of cell division to produce pre-B-cells where

IgL rearrangements take place.

Light chains come in two varieties, kappa (j) and lambda

(k), in mice and humans. In pre-B cells, Igj rearrangements

occur first, followed by Igk rearrangements. Since all pre-B

cells contain IgH proteins, successful production Igj or Igk

allows IgH ⁄ L-containing B-cell receptors (BCRs) to be

expressed on the cell surface. These cells are called immature

B cells. If the BCR on the surface of an immature B-cell in the

bone marrow recognizes self-antigen, receptor editing occurs,

which leads to replacement of the light chain gene variable

region (5, 6). Association of the new light chain with the

existing IgH chain may, or may not, change receptor speci-

ficity away from self-reactivity. If it does, recombination

ceases and the immature B cell can migrate to the periph-

ery. Immature B-cells which remain self-reactive despite

consecutive VL replacements are eliminated by apoptosis. Of

the approximately 18 million immature B cells that are pro-

duced daily in mice, it has been estimated that approxi-

mately 1–2 million migrate to peripheral lymphoid organs,

such as the spleen. It seems plausible that some form of

selection determines which immature B cells leave the bone

marrow.

Immature B cells that reach the spleen undergo further dif-

ferentiation to produce mature B cells that are competent to

mount immune responses (7, 8). Two fundamental changes

accompany peripheral B-cell differentiation. First, the func-

tional consequences of BCR crosslinking are altered. Newly

arrived immature B cells in the spleen, like their counterparts

in the bone marrow, die in response to BCR crosslinking. This

allows immature B cells to ‘checkout’ the peripheral environ-

ment for potential self-reactivity. An important difference

between antigen recognition by immature B cells in the spleen

versus the bone marrow is that receptor editing does not

occur in the spleen. During transition from immature to

mature B cells, the response to BCR-crosslinking changes from

cell death to cell proliferation. Second, mature B cells become

Fig. 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.

Kaileh & Sen Æ NF-jB function in B lymphocytes

Published 2012. This article is a US Government work and is in the public domain in the USAImmunological Reviews 246/2012 255

responsive to the survival cytokine BAFF (also known as BLyS)

which is essential for B-cell homeostasis.

Peripheral differentiation produces two major mature B-

cell subsets: (i) mature marginal zone (MZ) B cells that can

mount T-dependent and T-independent immune responses,

and (ii) follicular (FO) B cells that are the major source of

T-dependent immunity and B-cell memory. In addition, a

third subset of mature B cells, known as B1 B cells, are

present in the periphery; these are believed to be of fetal

origin (8). Here we review the role of NF-jB proteins in B-

cell development and function. Because this area of research

has been reviewed before (9, 10), we highlight the possi-

ble mechanisms involved and interesting interpretations of

the data, with a goal towards identifying areas for future

study.

NF-jB proteins in B-cell development

NF-jB proteins were identified based on binding to a highly

conserved sequence referred to as the jB site within the j

light chain gene enhancer (ijE) (11). Presence of NF-jB DNA

binding activity only in B lineage cells that transcribed IgL

genes, the demonstration that the jB element was important

for transcriptional activity of ijE (12) and later evidence of

the role of ijE in Igj gene rearrangements (13, 14) suggested

a role for NF-jB transcription factor in j gene activation dur-

ing B-cell development. The first indication that this was not

true came from the analysis of Nfkb1 gene deleted mice, which

lacked the p50 component of the ‘classical’ p50 ⁄p65 NF-jB

heterodimer (15–17). B-cell development and j gene expres-

sion was unaffected in these mice (18) calling into question

the prevailing hypothesis. Subsequently, p65 (RelA)-defi-

ciency was also shown to not affect B-cell development (19).

The double-deficiency of p50 and RelA was examined by

reconstituting fetal liver cells of the appropriate genotypes

into irradiated mice (20). In these experiments p50 ⁄RelA pre-

cursors generated B cells, but only in the presence of wild type

cells. While demonstrating that p50 ⁄ RelA NF-jB was not

essential for B-cell development (and by inference, Igj rear-

rangements), these observations also pointed towards an

essential cell non-autonomous NF-jB-dependent function

provided by hematopoietic cells for B-cell development. Based

on a very similar phonotype of IjB kinase 2 (IKK2)-deficient

precursors, Karin and colleagues proposed that systemic TNFa

produced during hematopoeitic reconstitution killed off T cell

precursors that were unable to induce NF-jB (21). This effect

was reduced when wild-type cells differentiated in the

same milieu as the mutant cells. Perhaps the lack of B-cell

production by p50 ⁄ RelA double-deficient precursors is caused

by a similar systemic stress response. While the simplest inter-

pretation of the synergistic effect of the double-deletion is that

p50 and RELA work together as NF-jB, it is also possible that

alternative functions of the p105 precursor are involved in

promoting cell survival during differentiation. Finally, Inlay

et al. (22) performed the key experiment of generating mice

with mutations in the jB site of ijE. Igj rearrangement, de-

methylation and expression was unchanged on the mutant

allele, thereby providing incontrovertible evidence that the

ijE jB site was not essential for j gene expression during nor-

mal B-cell development.

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

is that the ijE jB site regulates the developmental timing of

the j gene rearrangements in the bone marrow. During differ-

entiation, IgH gene rearrangements occur first at the pro-B cell

stage followed by Igj (or k) rearrangements at the subsequent

pre-B-cell stage. Functionally, the order of rearrangements

ensures that each IgH chain that is generated pairs with multi-

ple light chains to increase B-cell repertoire diversity. How-

ever, recent studies show that the j locus is in a permissive

nuclear environment for recombination at the pro-B-cell stage

(24, 25); yet, j transcription and the bulk of j recombination

is restricted to the pre-B-cell stage. Taken together with the

observation of low levels of j recombination in pro-B cells

(26, 27), one possibility is that p50 homodimer binding to

the jB site may suppress recombination in pro-B cells. Relief

of suppression in pre-B cells would permit robust recombina-

tion to occur. In this scenario, mutation of the jB site is

predicted to simply increase the level of j rearrangement in

pro-B-cells. Since IgH rearrangements would occur simulta-

neously, such a timing defect may not be reflected in grossly

disrupted B-cell development. Rather, alteration of the order

of rearrangements could be reflected more significantly in

the diversity of the B-cell repertoire that is generated. Along

the same conceptual lines, the ijE jB site may fine tune j

gene expression in mature B cells. For example, p50 homo-

dimers bound the jB site could reduce j gene transcription in

resting B cells by recruiting histone de-acetylases that are

associated with gene repression (28). During inflammatory

responses, p50 homo-dimers would be replaced by transcrip-

tion activating p50 ⁄ p65 or p50 ⁄ Rel heterodimers to enhance

j gene expression required to secrete large amounts of anti-

bodies of defined specificity.



Normal recombination order produces pro-B cells that

express IgH chains but not IgL chains. Association of IgH

chain with non-rearranging k5 and V-pre-B polypeptides

(surrogate light chains) expressed in pro-B cells generates a

signaling competent pre-B-cell receptor (pre-BCR). The pre-

BCR rescues pro-B cells from programmed cell death, induces

proliferation and differentiation to the pre-B-cell stage. The

significant similarities in signaling pathways initiated at the

pre-BCR and the BCR (29, 30) have suggested that the pre-

BCR activates NF-jB during the pro-B to pre-B-cell transition.

Indeed, use of NF-jB reporter mice (31) as well as direct

DNA binding assays (32) demonstrate higher levels of

transcriptionally competent NF-jB in pre-B cells compared to

pro-B cells. Recently a role for ATM, downstream recombina-

tion-induced DNA double strand breaks, has been proposed as

an NF-jB-inducing stimulus (33). How such a signal coordi-

nates with the pre-BCR-dependent developmental cues

remains unclear.

Yet, there is little genetic evidence for an important devel-

opmental role for NF-jB in the bone marrow. Other than the

p50 ⁄ RelA-double deficiency described above, none of the sin-

gle 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

or properties of pro- and pre-B cells (32, 36–38), despite

reducing NF-jB DNA binding activity in pre-B-cells. Interest-

ingly, modulating IjB function has been shown to affect early

B-cell differentiation. Specifically, ectopic expression of non-

degradable IjBa blocks transition of CD43+ pre-B cells to

CD43) pre-B-cells (31, 39), and the double-deletion of IjBa

and IjBe leads to substantial loss of pre- and pro-B cells in the

bone marrow (40). The increased apoptosis observed in pre-B

cells that express a non-degradable form of IjBa suggests an

anti-apoptotic role for pre-BCR-induced NF-jB, which can be

rescued by transgenic provision of Bcl-xL. There is evidence

that apoptosis in pro-B cells that lack effective IjB function

may be mediated by TNFa (41).

Despite the lack of obvious developmental defects in B lym-

phopoiesis, it is tempting to speculate on the possible roles of

pre-BCR-induced NF-jB besides survival. First, the genes IRF4

and 8 have been shown to be essential for appropriate devel-

opment to pre-B cells (42–44). IRF4) ⁄ IRF8) pre-BCR+ cells

continue to divide in response to IL-7 and do not initiate j

gene rearrangements. Because IRF-4 has been proposed to be

a NF-jB target gene (45), it may be one of the targets of

pre-BCR-induced NF-jB. In this way pre-BCR signaling would

initiate a self-limiting proliferative phase; cessation of cell

division would permit the activation of j gene rearrange-

ments. It is interesting to recall that onset of cellular quies-

cence has been related to induction of NF-jB and j gene

rearrangements in cell culture models as well. Specifically,

Abelson virus transformed pro- ⁄ pre-B-cell lines contain very

little nuclear NF-jB and do not express Igj. Induction of qui-

escence by turning off the Abl oncogene results in NF-jB

induction and j gene expression and rearrangements

(46–48). In these systems, blocking NF-jB induction reduces

j transcription and rearrangements. Another possible function

for pre-BCR induced NF-jB could be to induce chemokine

and chemokine receptor expression that would permit pre-

BCR-expressing cells to migrate to a different microenviron-

ment within the bone marrow. This may, in part, explain the

reduced numbers of pre- and pro-B cells in p100) ⁄ ) and

p50) ⁄RelB) mice (49, 50). Because such functions of pre-

BCR induced NF-jB are likely to enhance rather than play an

essential role in development, they may not be obvious in

steady-state analysis of bone marrow sub-compartments.

Alternatively, NF-jB functions at these stages may be mani-

fested at times of inflammatory stress or immune deficiency

when accelerated development would be advantageous.

NF-jB function in immature B cells in the bone marrow

Successful completion of light chain gene rearrangements

terminates V(D)J recombination at IgL loci and produces

immature B cells that express immunoglobulin (BCR) on the

cell surface. Cells expressing BCRs that are crosslinked by self-

antigens in the bone marrow (self-reactive BCRs) undergo

receptor editing (Fig. 1). This process attempts to alter BCR

specificity by re-activating V(D)J recombination at IgL loci to

generate new IgL chains to pair with the pre-existing IgH

chain. Because Igk rearrangements follow the cessation of Igj

rearrangements (51, 52), a substantial proportion of Igk-

expressing B cells in the mouse are produced as a consequence

of receptor editing. Several observations are consistent with a

role for NF-jB proteins at this stage of differentiation. First,

NF-jB DNA binding activity can be detected in immature

B cells, particularly those that are engaged in receptor editing

(53, 54). Second, conditional deletion of NEMO, the double-

deletion of IKK1 and IKK2, or deletion of TRAF6 (32) results

in a lower frequency of k+ immature B cells. Third, double-

deletion of Nfkb1 and 2 genes leads to reduced numbers of

immature B cells (55).

Analysis of the NEMO-deleted mice showed that the reduc-

tion in k+ cells was not a consequence of reduced receptor

editing because recombinase (RAG) gene expression and

RAG-induced DNA breaks were unaffected in this strain (32).



Restoration of k+ cells by ectopic expression of a Bcl-2 trans-

gene suggested a role for NF-jB-dependent survival; however,

Bcl-xL expression was not affected in NEMO or IKK1 ⁄ 2-defi-

cient immature B cells. Instead, mRNA of another pro-survival

kinase, Pim2, was attenuated in the absence of IKK activity.

Pim2 is considered to be a target of the noncanonical (alter-

nate) NF-jB pathway (56) and implicated in the survival of

mature B cells in response to BAFF (57, 58). Thus, extended

survival during receptor editing may be mediated, in part, by

BAFF-dependent mechanisms. It is also possible that other

receptors and ligands activate the noncanonical NF-jB path-

way at this differentiation step. A model that incorporates

both canonical (classical) and noncanonical NF-jB pathways

would explain the requirement for p50 ⁄ p52 in immature

B cells as well as the role of TRAF6 in generating of k+ cells.

Consistent with a dual signaling model, recent studies show

that immature B cells, particularly those that express self-reac-

tive sIg, are BAFF-responsive in vitro (59).

Overall, the following model emerges from these observa-

tions. Immature B cells in the bone marrow that express

self-reactive BCRs activate classical NF-jB via a NEMO ⁄ IKK-

dependent pathway. One of the consequences is up regulation

of BAFF-receptor on these cells, making them more responsive

to BAFF-dependent survival signals. Extended survival of these

cells permits continual receptor editing that is required to

generate k+ immature B cells. Disruption of canonical NF-jB

activation could affect cellular longevity by making cells less

sensitive to BAFF or by reducing levels of p100, an NF-jB tar-

get gene that serves as the substrate for the alternate NF-jB

pathway. As proposed by Derudders et al. (32), such short-

lived cells would initiate receptor editing but not be able to

proceed to ‘all the way’ to generate normal numbers of k+

immature B cells. Despite the requirement for NEMO or

IKK1 ⁄2, the signaling pathway from the BCR to NF-jB in

immature B cells remains unclear. In particular, k+ cell numbers

are unaffected in Bcl10-deficient mice (32, 60), leading to the

proposal that the BCR on immature B cells may signal to IKKs by

a CBM-independent pathway. Additional studies are required to

clarify how the BCR activates NEMO ⁄ IKK in these cells.

NF-jB function in peripheral B-cell differentiation

Immature B cells that arrive in the spleen are referred to as

transitional cells and require further differentiation into

mature functional B cells (7, 8) (Fig. 2). The most immature

transitional-1 (T1) cells are virtually indistinguishable from

immature B cells in the bone marrow with regard to function

and expression of cell surface markers. They are characterized

by extreme susceptibility to apoptosis upon BCR stimulation,

making them the target of negative selection against self-reac-

tivity in the periphery. BCR-induced cell death is mediated by

the mitochondrial pathway utilizing the pro-apoptotic BH3-

only domain proteins Bak, Bax, and Bim (61–63). T1 cells

develop into mature B cells via an intermediate transitional-2

(T2) stage. Whether all T1 cells that arrive in the spleen differ-

entiate further is not clear. Indeed, a current view is that T1

cells are destined to die unless ‘positively selected’ for further

differentiation (64, 65).

A critical role for NF-jB proteins during T1 to T2 differentia-

tion is evident from several genetic mutations (Fig. 2). First,

Rel ⁄ RelA double-mutant fetal liver cells produce only T1 cells

after transfer to irradiated hosts (66, 67). The extreme sensitiv-

ity to apoptosis of the residual IgM+ cells in this genotype is

partially rescued by a Bcl2 transgene; however, differentiation

remains incomplete indicating that Rel proteins are required

for differentiation as well as cell survival. Consistent with the

idea that REL and RELA are required for differentiation to T2

cells, conditional deletion of either NEMO or the double dele-

tion of IKK1 and IKK2, also blocks differentiation to T2 cells

(32). These observations suggest that signal-induced activation

of classical NF-jB pathway proteins is essential for T2 differen-

tiation. The fact that single deletion of either Rel or RelA, or

either IKK gene, does not significantly impair differentiation

likely reflects compensatory activity of the remaining proteins.

The most likely source of NF-jB-induction in T1 cells is the

BCR. This is most clearly exemplified by conditional deletion

of CD79a, the BCR signal transducing module, in immature

B cells in vitro (68, 69). Absence of this protein leads to dra-

matic alteration of the gene expression profile in these cells

towards a pattern that is more similar to that seen in pro-B

cells. Thus, constitutive BCR signaling maintains the state of

T1 cells and is required for developmental progression. Addi-

tionally, deficiency of several cytoplasmic signaling molecules

that transduce BCR signals in mature cells also affect periph-

eral differentiation at transitional stages. The most prominent

among these are Btk, PLCc, PI3K and the adapter proteins

BLNK and BCAP; however, the effect of each mutation varies

(70–79). Thus, the case for BCR signaling in transitional

B cells is strong.

However, the case for BCR signaling to NF-jB is relatively

weak. The strongest evidence against this idea is that defi-

ciency in any of the CBM complex (Carma-1, Bcl10, and

Malt1) proteins does not affect peripheral B-cell differentiation

significantly (80–84). Because these proteins are essential for

BCR-induced NF-jB activation in mature B and T cells (85,

86), the lack of developmental phenotypes of CBM mutations



has been taken to indicate that BCR signaling to NF-jB is not

involved in transitional B cells. There is some evidence, how-

ever, that absence of Bcl10 adversely affects maturation of T2

cells to follicular B cells (60). Moreover, biochemical studies

also indicate that BCR crosslinking by anti-Ig does not induce

classical NF-jB DNA binding in T1 cells (87, 88). How can

we reconcile the requirement for inducible canonical NF-jB

components in T1 ⁄ T2 differentiation with the apparent

absence of a connection to the BCR? The most prosaic expla-

nation is that the NF-jB-inducing signal originates at a recep-

tor other than the BCR. While we cannot rule out this

possibility till such a receptor is identified, we consider it

more likely that the BCR in T1 cells is connected to IKKs dif-

ferently than it is in mature cells (89). Mechanistically, this

may be because the BCR in T1 cells is not associated with lipid

rafts (90–92), resulting in a distinct constellation of signaling

proteins in its vicinity.

Differentiation to the T2 transitional stage requires alternate

NF-jB function. This is exemplified by blocks at the T2 stage

of development in Nfkb1 and Nfkb2 double-deficient precursors

(55), as well as in IKK1-deficient precursors (56). The

response of T2 cells to BCR crosslinking is distinct from T1

cells in several ways that involve NF-jB proteins. First, BCR

crosslinking induces classical NF-jB and long-term REL induc-

tion in T2 cells (88). Both these are characteristic of the NF-

jB response of mature B cells (see below). Consequently, REL

target genes, such as Bcl-xL and A1, are activated in T2 cells

but not in T1 cells. This is likely to be an important mecha-

nism that makes T2 cells less susceptible to BCR-induced cell

death. The molecular basis for differential BCR signaling to

REL in T2 cells is not known. Second, BCR signals in T2 cells

upregulate expression of Nfkb2 gene resulting in the produc-

tion of p100 protein (88). This is potentially an important

aspect of the survival characteristics of T2 cells because p100

is an important substrate for BAFF-R-dependent survival

signaling (see below). Third, T2 cells begin express higher

levels of BAFF-R and become responsive to survival signaling

by BAFF. Two observations provide clues as to how BAFF-R

expression is regulated in the T1 to T2 transition: (i) BCR

crosslinking upregulates BAFF-R expression in mature B cells

(93), and (ii) this does not occur in Rel-deficient cells (88).

Thus, upregulation of BAFF-R in T2 cells may also reflect the

connection of BCR signaling to Rel in these cells. Overall, T2

cells bear a ‘more competent’ BCR and higher levels of BAFF-

R, which together may determine many of the characteristics

of these cells.

While development beyond T2 stage requires BAFF ⁄ BAFF-R

interactions, recent evidence indicates that such signals may

be initiated earlier in T1 cells. Specifically, Hoek et al. (94)

showed that B-cell development in mice deficient for both Btk

and BAFF-R was blocked at the T1 development stage. Since

each individual mutation blocks differentiation at the T2

stage, these observations suggested that some BAFF signaling

occurred at the earlier stage as well. Additionally, Rowland

et al. (59) showed that differentiation of immature bone mar-

row B cells to transitional cells was enhanced in the presence

of BAFF (T1 cells express BAFF-R, though at lower levels than

T2 cells). Perhaps the reduced importance of BAFF ⁄ BAFF-R at

the T1 stage is in part due to inefficient BCR signaling

that prevents BAFF-R upregulation or efficient generation of

p100.

NF-jB proteins in mature B-cell survival

NF-jB proteins have essential roles in the generation and ⁄or

maintenance of mature B cells. Of the two major subsets of B

cells produced in the adult, MZ B cells are more susceptible to

NF-jB deficiency. This is reflected in reduced MZ B cells

numbers in several Rel-family single gene deficiencies, includ-

ing Nfkb1 and RelB (Fig. 2). Absence of Rel appears not to affect

Fig. 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.



MZ B cells, and their status in RelA deficiency has yet to be

clearly defined. Follicular (FO) mature B cells are less affected

by single gene deficiencies, though they are significantly

reduced in several compound deficiencies such as Nfkb1 ⁄ 2,Nfkb1 ⁄ RelB, and RelA ⁄ Rel. Both kinds of mature B-cell subsets

are also reduced in mice that lack IKK1 and IKK2 or NEMO.

These observations demonstrate that both canonical and

noncanonical pathway NF-jB proteins are required for mature

B-cell generation and ⁄or maintenance, and that their function

must be induced via the IKKs.

Two receptors that are known to be essential for mature B-

cell survival are the BCR and the BAFF-R. Mice that carry

genetic mutations in genes encoding either BAFF or the BAFF-

R lack mature B cells (95, 96). BAFF ⁄BAFF-R stimulation is

required continuously to maintain the peripheral B-cell pool

since intravenous administration in mice of Fc receptor fusion

proteins that bind BAFF results in the rapid loss of mature B

cells (97, 98). The survival response of mature B cells to BAFF

requires a signaling-competent BCR. This was shown by con-

ditional deletion of genes encoding either the immunoglobu-

lin heavy chain (99) or the signal-transducing chaperones Iga

and Igb (100); disruption of the BCR complex led to loss of

mature B cells within 24–48 h, despite the presence of sys-

temic BAFF.

Because the BCR on naive B cells provides survival signals in

the absence of overt antigen, this kind of signaling has been

referred to as ‘tonic signaling’ (101). BCR signaling that

occurs in immature B cells (discussed above) is also a kind of

tonic signaling, though its similarity to survival signaling in

mature B cells remains unclear. While the term tonic signaling

implies that it occurs in the absence of BCR recognition, it is

quite possible that it is mediated by weak interactions of the

BCR with self-molecules whose affinity is below the threshold

to induce negative selection (at the immature stage) or to

trigger self-reactivity (at the mature stage). Rajewsky and

colleagues (102) have genetically explored the signaling

pathway downstream of tonic BCR signaling and found that

loss of the BCR on mature B cells can be compensated by

provision of a constitutively active form of the catalytic sub-

unit of PI3 kinase. In contrast, constitutively active IKK2,

constitutively active Akt or Bcl-2 did not rescue BCR defi-

ciency. The mechanism by which active PI3K permits B cells

to respond to BAFF signaling remains unclear. It is likely

that tonic BCR signaling is a source of constitutive NF-jB in

mature B cells via continued degradation of IjBa (see

below). While this may be an essential function, signaling

to NF-jB does not recapitulate all functions of tonic BCR

signaling.

BAFF ⁄ BAFF-R activates canonical and noncanonical

NF-jB

BAFF ⁄BAFF-R interaction activates multiple pro-survival

mechanisms in mature B cells, including activation of the non-

canonical NF-jB pathway (103, 104). In accordance with an

important role for this pathway in B-cell survival, Nfkb2-defi-

cient B cells are refractory to BAFF-dependent survival in vitro.

However, the presence of mature follicular B cells in

Nfkb2- and RelB-deficient mice (105, 106) indicates that other

features of BAFF-R signals compensate for the lack of these

proteins in vivo. One of the prominent nuclear targets of the

resulting p52 ⁄ RelB heterodimer is the gene encoding the

Ser ⁄ Thr kinase Pim2 (56). Unlike Nfkb2-deficient B cells, how-

ever, Pim2-deficient B cells respond to BAFF treatment in vitro

with increased viability indicating that p52 ⁄RelB must activate

additional survival genes in BAFF-treated cells (58). These

currently unknown genes appear to be downstream of mTOR

because BAFF-responsiveness of Pim2-deficient cells is abro-

gated by rapamycin. Woodland et al. (58) connected BAFF-

dependent survival signaling via mTOR and PIM2 to the

induction ⁄ maintenance of Mcl-1 expression. Though the

mechanism of Mcl-1 induction ⁄maintenance is not clear, this

is an important connection because, along with components

of the BCR or BAFF-R and BAFF, Mcl-1 is essential for survival

of mature B cells in mice (107).

In addition to the noncanonical NF-jB pathway, BAFF ⁄BAFF-R interaction has also been shown to activate canonical

NF-jB via a Btk-dependent pathway, resulting in induction of

RELA- and REL-containing DNA binding activities (108, 109).

This induction is relatively rapid and may provide short-term

survival function to B cells treated with BAFF. However, long-

term survival in the presence of BAFF requires kinases and

proteins of the noncanonical NF-jB pathway. Short-term

canonical NF-jB activation by BAFF may also contribute to

BAFF-dependent survival by inducing p100 protein to serve as

a substrate for the noncanonical pathway. In this way, BAFF

treatment can initiate a positive autoregulatory loop involving

canonical and noncanonical NF-jB activation. However, the

loop is not sufficient by itself to induce long-term BAFF-

dependent B-cell survival in the absence of the BCR.

Despite the critical role of the noncanonical NF-jB pathway

in mediating BAFF-dependent survival signals, expression of

constitutively active IKK2 (Ikk2ca), which induces canonical

NF-jB, in mice completely restores the B-cell deficiency in

BAFF-R-deficient mice (110). This includes generation of fol-

licular mature and marginal zone B cells and restoration of

splenic architecture. B cells from Ikk2ca mice have higher



nuclear levels of classical NF-jB proteins, such as RELA, but

not of p52, suggesting that generation and survival of mature

B cells in these animals is entirely dependent on canonical NF-

jB pathway signaling. These observations suggest that synergy

between BCR and BAFF-R for B-cell survival may ultimately

funnel through activation of classical NF-jB. Perhaps the con-

stitutive NF-jB DNA binding activity that is present in human

and mouse mature B cells reflects canonical pathway activation

by both these receptors.

Cross-talk between BCR and BAFF-R

Regulating B-cell homeostasis via two receptors provides flexi-

bility in modulating survival requirements during different

phases of the immune response. For this, it is imperative that

the two receptors cross-talk. This occurs at multiple levels

between BAFF-R and the BCR. As briefly described in preced-

ing section, BCR crosslinking on mature cells increases expres-

sion of the BAFF-R, thereby sensitizing cells to BAFF

signaling. In addition, NF-jB activation by of the BCR acti-

vates Nfkb2 and RelB gene expression (111–113), which are

substrates for alternate pathway activation by BAFF-R. We

have proposed that activation of p100 as a consequence of

tonic BCR signaling may be an important determinant for the

dual requirement of the BCR and BAFF-R for maintenance of

mature B cells (114). In these studies, pharmacological inhibi-

tion of Syk tyrosine kinase activity in mature B cells ex vivo

abrogated BCR ligand-independent upregulation of p100. Syk

inhibitor-treated cells did not maintain long-term alternate

NF-jB activation in response to BAFF, which correlated with

reduced survival of these cells. Our interpretation of these

observations is that continuous p100 protein production in

response to BCR-initiated signals is essential for BAFF-depen-

dent cell survival. In follow up studies, we found that PI3K

activity is required for tonic, or BCR-inducible, expression of

p100 (unpublished data, MK and RS). Thus, one way in

which PI3K activity may compensate for tonic BCR signals is

by generating a pool of p100 protein that can mediate BAFF-

R-initiated survival signals.

Conversely, BAFF-R-initiated signals enhance BCR signals in

several ways. For example, BAFF ⁄BAFF-R interaction induces

PI3K and Akt activation (58, 115, 116). PI3K activity has been

shown to be essential for BCR-induced NF-jB activation in B

cells (117), and active forms of Akt have been shown to

induce NF-jB-dependent transcription in reporter assays

(118–120). Continuous in vivo stimulation of B cells with

BAFF may produce constitutively high basal PI3K activity in

these cells that lowers the threshold of tonic BCR signaling to

NF-jB. Additionally, high basal PI3K activity may enhance

responses to NF-jB-inducing stimuli during immune

responses. The continuous requirement for PI3K activity in B

cells is exemplified by the absence of mature B cells in mice

deficient in various components of PI3K (74, 77, 121), as

well as the extreme sensitivity of mature B cells to apoptosis

upon pharmacologic inhibition of PI3K ex vivo. However, these

components have not been conditionally deleted after B-cell

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

CD21, a component of the CD19 ⁄ CD21 co-receptor complex

(122, 123). This complex lowers the threshold for BCR sig-

naling thereby completing another mutually re-enforcing

loop, whereby signals through the BCR feed into the BAFF-R

pathway and, conversely, BAFF-R signals enhance BCR signal-

ing. Each of these loops involves components of canonical and

noncanonical pathway NF-jB proteins.

NF-jB function in mature B cells

Beyond the essential requirement for NF-jB proteins for B-cell

maturation and homeostasis, inducible NF-jB activation is

critical for effective immune responses. The major initiators of

NF-jB signaling in B cells are the BCR, various members of

the TNF receptor superfamily (in particular BAFF-R, TACI,

BCMA and CD40), and Toll-like receptors (in particular TLR4

and 9). The constitutive nuclear NF-jB found in murine sple-

nic B cells consists primarily of REL-containing complexes

(124–126); the hetero-dimeric partner is most likely p50,

though the presence of B cells in Nfkb1-null mice indicates that

p52 can substitute effectively or that REL homodimers suffice

in the absence of p50. Similarly, REL and RELA also have

mutually compensatory functions because genetic deletion of

either does not impair the generation of mature B-cells. We

have proposed that the predominance of REL-containing com-

plexes in B cells may be due to increased nuclear export of

RELA-containing complexes by IjBa because of the nuclear

export sequence (NES) present at the C-terminus of RELA

(127–130). REL does not contain a corresponding NES and

may therefore be better retained in the nucleus compared to

RELA. Indeed, B-cell differentiation and function is hampered

in mice whose IjBa-protein lacks an NES (131). In these mice

non-functional REL ⁄ IjBa- complexes accumulate in B-cell

nuclei, making them unavailable for IKK-dependent activation.

This leads to reduced Nfkb2 and RelB gene expression and, as a

consequence, both canonical and noncanonical NF-jB induc-

tion and function is impaired. These observations show that



shuttling of NF-jB proteins between the nucleus and

cytoplasm is an essential functional feature of these proteins,

and highlight the connection between canonical and noncano-

nical NF-jB signaling in the maintenance of mature B cells.

NF-jB response to BCR crosslinking

BCR crosslinking with anti-l chain F(ab¢)2 fragment has been

used as a surrogate for antigen-dependent B-cell activation.

This treatment leads to rapid NF-jB nuclear translocation via

the canonical pathway. The signaling pathway that connects

the BCR to NF-jB has been intensely studied and summarized

in several excellent reviews (10, 132). Briefly, BCR crosslink-

ing activates Src tyrosine kinases leading to phosphorylation

of ITAM motifs in BCR-associated signaling proteins CD79a

and b (Iga and Igb). These phosphorylated ITAMs recruit and

thereby activate the Syk kinase, which via phosphorylation of

adapter proteins such as BLNK, Bam32, and BCAP results in

the activation of downstream kinases cascades including

MAPKs and PI3K. Syk also activates Btk, which is essential for

phosphorylating and activating PLCc. PLCc enzymatic activity

results in the generation of IP3 and diacyl glycerol; the former

binds to receptors in the endoplasmic reticulum (ER) to

release calcium from ER stores and the latter is required to

activate protein kinase C b (PKCb). PKCb phosphorylates the

adapter CARMA1 leading to generation of a complex contain-

ing CARMA1, Malt1 and Bcl10 (the CBM complex) which

serves as a scaffold to bring together the IjB kinases and the

kinase that activates IKKs. In B cells the latter is likely to be the

TGFb-activated kinase 1 (TAK1). TAK1-mediated phosphory-

lation of IKK2 results in IKK2 activation, which then phospho-

rylates IjB proteins leading to their ubiquitination and

degradation. As a consequence NF-jB proteins, that were

bound to the IjBs are free to translocate to the nucleus and

activate gene expression. This multi-enzyme cascade leads to

NF-jB activation within 30 min of BCR crosslinking in naive

murine splenic B cells; the resulting nuclear NF-jB consists of

RELA- and REL-containing homo- and heterodimers.

Despite the considerable detail in which this pathway is

understood, some features remain unclear. One of these is the

intriguing observation that the characteristics of B cells singly

deficient in either CARMA1, or Bcl10 or Malt1 are not

identical with regard to NF-jB activation. In particular, Bcl10-

deficient B cells do not activate IKKs (and thereby do not

induce NF-jB) in response to BCR crosslinking whereas Malt1-

deficient B cells activate IKKs (though not as robustly as

wildtype B cells) leading to IjB degradation (133). However,

nuclear NF-jB that is induced in Malt1-deficient B cells is

significantly diminished in REL-containing complexes. Based

on additional biochemical studies, Ferch et al. (133) con-

cluded that presence of MALT1 in the WT CBM complex tar-

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

observation is the existence of another level of molecular rec-

ognition that distinguishes RELA- or REL complexes. This

could occur, for example, by spatial segregation of RELA- or

REL complexes within the cytosol such that active IKKs need

to be directed to different subcellular regions to activate each

complex. Alternatively, RELA- or REL-containing complexes

may need to be recruited to distinct compartments for the

associated IjBs to be phosphorylated by IKK; in this model,

the sub-compartments that contain CB or CBM complexes

may be different. Finally, it is possible that REL and RELA are

differentially associated with IjBa, b, and e. In this scenario,

‘weakened’ NF-jB signaling via the CB complex may target

one IjB better than the others, leading to induction of the Rel

proteins associated with that IjB but not the other IjBs. Some

evidence for differential REL ⁄ IjB association and its functional

consequences have been previously noted in T cells (134).

Spatial control may also explain the essential requirement

for PI3K in canonical NF-jB activation via the BCR. Since dele-

tion of genes encoding PI3K catalytic or regulatory subunits

lead to developmental defects that prevent generation of

mature B cells, the best evidence of a role for PI3K in NF-jB

activation comes from pharmacologic inhibition of PI3K dur-

ing BCR crosslinking of mature wildtype cells (117). The most

likely point of intersection of PI3K with the signaling scheme

summarized above is Btk. Reduced NF-jB induction in B cells

from xid mice (135, 136), that express Btk protein with a

point mutation in the membrane-targeting PH domain, has

been attributed to lack of Btk activity. However, recent studies

show that Btk activation, as evidenced by production of phos-

pho-Btk is normal in xid B cells. The problem seems to be that

xid Btk is unstable and present at low levels in the cells, sug-

gesting that membrane recruitment by interaction with PIP3

stabilizes the protein (137). Thus, PI3K-dependent PIP3 pro-

duction is a key intermediate in maintaining sufficient levels

of Btk. Additionally, PLCc activation by membrane-bound Btk

may localize active PLCc close to its substrate in the plasma

membrane for effective function. In this manner, membrane

localization of Btk and PLCc may drive NF-jB activation in

B cells. It is interesting to note that NF-jB activation in T cells

also requires PI3K, though for different reasons. In T cells,

non-classical PKCh is the enzyme that activates the CBM

complex by phosphorylating CARMA1. PKCh activation is



mediated by the kinase PDK1 which, like Btk, contains a PH

domain and requires binding to PI3K-dependent PIP3 for

activity (138). PI3K activity in T cells requires co-crosslinking

of the T-cell antigen receptor and the co-receptor CD28; in

B cells PI3K may be activated by BCR-associated CD19 (139,

140) or activation of the adapter protein BCAP after BCR

crosslinking (141, 142).

Kinetics of NF-jB activation by BCR

NF-jB induced by the canonical pathway in response to BCR

crosslinking is transient (143). This wave of NF-jB contains

both RELA- and REL-containing DNA-binding proteins,

reaches a maximum at 1–2 h post-activation and is consider-

ably reduced by 6 h. Thereafter, nuclear RELA levels remain

low despite continued presence of BCR crosslinking antibody.

At longer time points lasting until 24 h the NF-jB response is

dominated by REL. We refer to these as Phases I and II of NF-

jB induction and have proposed that each serves distinct func-

tions. These observations raise two questions: (i) what is the

mechanism that restricts classical NF-jB to one cycle of activa-

tion, and (ii) what are the functions of each phase?

Several possible mechanisms have been put forward for lim-

iting the duration of NF-jB activation in various cell types and

in response to diverse NF-jB activators (144). However, most

of these mechanisms have not been experimentally evaluated

in BCR-activated B cells. Our working model is that the major

contributor to Phase I NF-jB downregulation is repression

mediated by newly-synthesized IjBa. This is the oldest model

of post-activation NF-jB suppression (145, 146) and is based

on NF-jB-dependent transcription and de novo synthesis of

IjBa protein. The newly synthesized IjBa migrates into the

nucleus, removes DNA-bound IjBa and exports it out of the

nucleus (130, 147, 148). The cytosolic NF-jB ⁄ IjB complex

may not be re-induced despite continuous BCR crosslinking

for several reasons. One possibility is that re-expression of sur-

face Ig after receptor endocytosis takes substantially longer

(149, 150) than the duration of phase I NF-jB. Moreover,

continued BCR crosslinking and resulting re-endocytosis may

prevent expression of substantial levels of the BCR to permit

effective IKK activation to induce a second cycle of canonical

NF-jB activation. Additional mechanisms may also reduce the

effectiveness of BCR signaling to NF-jB, such as inactivation

of Bcl10 by degradation or phosphorylation as has been noted

in T cells (151, 152), or activation of de-ubiquitinating

enzymes such as A20 and CYLD (153). An important role for

A20 in B-cell physiology is evident from the observation that

B-cell-specific A20 deficiency leads to hyper proliferation and

autoimmunity, together with elevated expression of NF-jB

target genes (154–156). However, the stage at which B-cell

activation is most susceptible to A20-dependent downregula-

tion remains unclear. Similarly CYLD-deficiency results in

higher basal levels of NF-jB in B cells due to higher IKK2

activity and increased numbers of MZ B cells (157, 158).

Function of Phase I NF-jB

Identifying target genes of a specific transcription factor

requires a combination of assays which must include: (i) eval-

uation of transcript levels in the presence, or absence, of the

transcription factor, (ii) chromatin immunoprecipitation to

determine transcription factor binding to important regulatory

sequences of putative target genes, and (iii) evaluation of the

importance of the identified binding sites for gene transcrip-

tion. NF-jB targets in activated B cells have not been indenti-

fied in this comprehensive fashion; however, the identity of

some putative NF-jB targets affords a perspective into the

function of phase I NF-jB. Amongst genes that were highly

induced within the first 3 h were the chemokines CCL3 and

CCL4, the chemokine receptor CCR7, the transcription factors

IRF4 and c-Myc and the signaling proteins DUSP1 and Plk3.

CCL3 and 4 are interesting because they serve as chemoattrac-

tants for CD4+ T cells (159). By attracting CD4+ T cells, anti-

gen exposure increases the probability of B cells to present

antigens to T cells of the right specificity. Once activated such

T cells would provide help to B cells via the CD40 ⁄CD40L

pathway. The chemokine receptor CCR7 has been implicated

in the movement of B cells towards the T-cell zone in the

spleen (160), which is enriched for CCL21, the chemokine

ligand of CCR7. We have proposed that these Phase I NF-jB

genes would maximize the possibility of B ⁄T encounter to ini-

tiate T-dependent B-cell immune responses. The transcription

factor c-Myc is essential for G1 progression of activated B cells

(161, 162), and IRF4 is known to be required for cell growth

and differentiation of B cells (163). Thus, putative NF-jB tar-

get genes induced during Phase I serve a range of functions

including re-distribution of B cells, inducing cell cycle pro-

gression and differentiation, and altering signal transducing

properties.

One of the ways we imagine that T-dependent immune

responses are initiated is antigen binding to the BCR, followed

by endocytosis, proteolytic digestion, and expression of anti-

genic peptides on MHC class II molecules on the cell surface.

These MHC class II-bound peptides are recognized by T cells

of the appropriate specificity, leading to activation of antigen-

specific T cells that provide B-cell help. To mimic this



scenario, where there is limited BCR engagement with antigen,

we used a pulse-activation protocol to stimulate B-cells with

single round of surface BCR signaling. Virtually the entire

phase I NF-jB program was recapitulated with this form of

pulse BCR-activation. Nuclear RELA and REL proteins were

induced with indistinguishable kinetics in pulse- versus con-

tinuously activated cells, and gene expression analysis in

pulse- or continuously activated cells showed that 70–80% of

inducible genes were comparably induced during the period

of phase I NF-jB activation under both conditions (143).

These included 80% of putative NF-jB target genes induced

over the course of the first 3 h, among which were genes

highlighted above. Despite normal up- and down-regulation

of c-Myc mRNA, however, pulse-activated B cells did not

show any evidence of G1 progression. Instead, these cells

responded more robustly to CD40 crosslinking as evidenced

by biochemical markers of G1 progression such as Cdk2 ⁄4and Cyclin D2 ⁄ E expression, and phosphorylation of retino-

blastoma protein. Additionally anti-CD40 treatment of pulse-

activated B cells resulted in a more rapid increase of cell size

compared to naive B cells activated by CD40. Taken together,

with activation of CCL3, CCL4 and CCR7 our working model

is that pulse-activation primes B cells in different ways to

receive T-cell help.

Characteristics of phase II NF-jB

Phase II NF-jB, which requires continuous BCR crosslinking,

is dominated by nuclear expression of REL and coincides with

de novo Rel transcription and translation. Early studies showed

that long-term survival of BCR-activated cells was severely

impaired in Rel-deficient B cells due to reduced expression of

the anti-apoptotic genes Bcl-xL and A1 (164, 165). These

observations are almost entirely the results of Phase II Rel

activation. Accordingly, Bcl-xL is transiently expressed in

pulse-activated cells, while A1 expression requires continuous

treatment with anti-IgM. Thus, a major function of Phase II

NF-jB is to maintain cell viability in BCR-activated cells in

order to permit cell division. Rel is known to be an inducible

gene and has been proposed to be auto-regulated by NF-jB.

Our preliminary results show that phase II Rel induction

occurs normally in PKCb-deficient B cells where classical NF-

jB activation is impaired, and in WT B cells activated in the

presence of a pharmacologic inhibitor of IKK2. These observa-

tions indicate that canonical NF-jB induction is not required

to induce phase II REL. Instead, Rel expression is sensitive to

calcium chelators and is blocked by cyclosporine A (CsA)

treatment during BCR activation (166, 167). Sensitivity to

CsA points to two well-studied transcription factors, NF-AT

and Mef2c, whose nuclear induction and ⁄or transcriptional

activity are suppressed by CsA (168–170). Though phenotyp-

ically Mef2c-deficient B cells resemble Rel-deficient B cells in

terms of their sensitivity to BCR-induced death and lack of

BclxL mRNA induction (171), REL induction in response

to anti-IgM occurred normally in Mef2c-deficient B cells.

Though Rel induction has not yet been directly analyzed in

NF-AT-deficient mice, Gerondakis and colleagues (172) have

previously proposed that Rel mRNA in TCR-activated T cells is

induced by NF-AT. Our working hypothesis is that Rel gene

induction by the BCR is also mediated by NF-AT proteins.

NF-jB response to CD40

T-dependent immune responses generate different classes of

high affinity antibodies via class switch recombination (CSR)

and somatic hypermutation (SHM). These processes which

occur in germinal centers within the spleen and Peyer’s

patches within the gut, critically require CD40 on the B-cell

surface and induced CD40 ligand (CD40L) on CD4+ T-

follicular helper cells. CD40 or CD40L deficiency in mice leads

to defective affinity maturation of antibody responses, and

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-

tion of naive B cells by CD40 crosslinking, or via CD40L, leads

to cell proliferation and CSR; signaling to NF-jB is essential

for these functions of CD40.

Like other members of the TNF-receptor superfamily, CD40

signals canonical NF-jB induction by activating IKK complex

via TRAF proteins (173, 174). One of the most significant dif-

ferences between CD40-induced and BCR-induced canonical

NF-jB is that unlike the BCR, CD40 induces persistent NF-jB

activation (175). This NF-jB comprises of both RELA- and

REL-containing DNA-binding activities. The basis for persistent

NF-jB activation by CD40 has not been satisfactorily explained.

Like the mechanism proposed for persistent NF-jB activation

by LPS in mouse embryo fibroblasts (176, 177), one possibility

is that CD40 crosslinking produces an NF-jB-inducing cyto-

kine that feeds back to reactivate NF-jB in these cells. However,

such a cytokine has not been identified. Alternatively, it is pos-

sible that downregulatory mechanisms, such as degradation of

intermediate cytosolic signaling proteins, are not efficiently

activated after CD40 stimulation. In this scenario, post-activa-

tion repression by de novo synthesized IjBa may be ineffective

because the rate of IjBa degradation (by continued CD40 sig-

naling) out-competes the rate of new IjBa synthesis.



Despite persistent induction of RELA, Rel-deficient B cells do

not proliferate or carry out CSR in response to CD40. Thus,

RELA apparently cannot substitute for some essential func-

tion(s) of REL. The transcription factors E2F3, Myc, and IRF4

have been proposed to be Rel-responsive target genes involved

in the proliferative response; whether these genes are suffi-

cient to explain the lack of proliferation of Rel) ⁄ ) B cells

remains to be determined. The critical role of REL in mediat-

ing CD40 signals is also emphasized by the lack of organized

germinal center formation after immunization of Rel-deficient

mice with T-dependent antigens as well as reduced CSR in vivo

and in vitro (178). This effect is exacerbated in p50 ⁄ REL dou-

ble-deficient mice. It is interesting that CD40 treatment of B

cells does not induce the equivalent of Phase II REL that is

mediated by de novo Rel transcription and translation. This is

consistent with the observation that Rel transcription depends

on a CsA-sensitive pathway, which is not activated by CD40.

Thus, the proliferative function of REL in CD40-treated cells is

mediated entirely via IKK activation.

Stimulation via CD40 also activates the alternate NF-jB

pathway via recruitment of TRAFs 2 and 3 leading to p100

degradation and release of p52 ⁄ RelB to activate transcription.

Whereas BAFF-R-induced p52 is essential for B-cell survival

ex vivo, CD40-induced p52 is not required for cell viability

(175). The major function of the pathway may be to induce

chemokine genes that are required for generation and mainte-

nance of germinal centers. An important difference between

CD40 and BAFF-R with regard to alternate NF-jB activation is

that CD40 efficiently induces expression of p100 whereas

BAFF-R does not. By constantly replenishing the store of p100

required to generate p52, CD40 stimulation is able to main-

tain long-term noncanonical NF-jB activity. p100 upregula-

tion is presumably due to sustained classical NF-jB induction

by CD40, but this has not been established yet. The close

working relationship between the canonical and noncanonical

NF-jB pathways in response to CD40 is exemplified by identi-

fication of hypomorphic mutations in NEMO that cause

hyper-IgM syndrome in humans (179) that has a phenotype

very similar to deficiency of CD40L (and thereby loss of all

CD40-dependent signaling). Jain et al. (179) showed that B

cells from these patients induce classical NF-jB poorly, and lack

somatic mutations and class switched Ig genes. Thus, loss of the

classical pathway alone is sufficient to impair CD40 function.

NF-jB response to LPS

Bacterial lipopolysaccharide was the first identified NF-jB-

inducing agent (180). The close correlation between

LPS-induced NF-jB DNA binding and j gene transcription in

pre-B-cell lines was the basis of the idea that NF-jB directly

activated j gene transcription via the j intron enhancer. More-

over, ‘super-induction’ of NF-jB DNA binding activity by LPS

in the presence of protein translational inhibitors led to the

post-translational model (180) that is currently referred to as

the canonical pathway. Super-induction of NF-jB also pre-

saged the idea of post-induction repression by a newly synthe-

sized inhibitor of NF-jB. LPS was used in these early studies

because of its well-known property of being a B-cell mitogen.

The current state of the mature B-cell response to LPS has

been recently reviewed (181). LPS treatment induces REL-

and RELA-containing NF-jB persistently via the canonical

pathway; the noncanonical NF-jB pathway is not activated by

LPS. LPS-induced proliferation is significantly reduced in B

cells that lack Nfkb1 or Rel, though G1 progression occurs nor-

mally. NFkb1- and Rel-deficient cells are also more sensitive to

apoptosis after LPS treatment, and this effect is heightened

considerably in Nfkb1 ⁄ Rel double-deficient cells. These obser-

vations suggest that viability of LPS-activated B cells in syner-

gistically maintained by these two factors. Gerondakis and

colleagues (182) have provided a plausible mechanism for the

cooperative effects of NFKB1 and REL on B-cell survival. They

showed that NFKB1-associated Tpl1 kinase activates the ERK

pathway in LPS-treated cells. Active ERK phosphorylates the

pro-apoptotic protein Bim leading to its degradation. Concur-

rently, LPS-induced nuclear REL activates transcription of anti-

apoptotic Bcl-XL and A1 gene expression, which neutralize

the activity of residual Bim by direct interactions. Thus, in

wildtype B cells reduced Bim and elevated Bcl-XL ⁄ A1 together

maintain cell viability. Absence of one of the survival path-

ways in either of the single gene deficiencies results in partial

sensitivity to cell death, while the absence of both survival

pathways makes Nfkb1 ⁄ Rel double-deficient cells super-sensi-

tive to apoptosis. Beyond maintaining cell viability, Rel is also

essential for S-phase entry of LPS-treated B cells.

Perspectives: conclusions and outstanding questions

Through the work of many scientists the influence of NF-jB

now extends well beyond its originally proposed role as a j

gene activating transcription factor that is important for B-cell

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

continuing to yield insights into B-cell biology. The most ele-

gant advances that have been made in the NF-jB field are the

delineation of signaling pathways that connect diverse cellular



stimuli to NF-jB. Identification of the components involved

and their subsequent genetic manipulation have revealed

many biological situations that utilize NF-jB. Biochemical

studies of how these components function have provided tar-

gets for therapeutic intervention. An area that appears ready

for equally sophisticated analyses is the regulation of gene

transcription by NF-jB.

At the simplest level the function of the jB site in the j

enhancer remains mysterious. In this review, we have hypoth-

esized possible functions for this site based on the idea that its

high conservation between species must serve a purpose.

Additional studies are necessary to confirm or refute these

conjectures. Moreover, little is known about cooperation

between the jB site and other protein binding sites within the

enhancer. Obviously, this functional question cannot be

addressed till we understand the function of the jB site itself.

A broader, more open-ended, question pertains to the sub-

unit-specific functions of Rel family proteins. It is noteworthy

that many of the functional studies have been carried out with

conditional deletions of canonical or noncanonical signal

transducing components rather than manipulating genes

encoding Rel family members. While these studies demon-

strate the importance of one or the other pathway, they do not

readily provide insight into biological phenomena based on

NF-jB-dependent gene expression. This applies particularly to

distinguishing between the overlapping functions of REL and

RELA. Additional complexity is introduced because REL- or

RELA-dependent transcription is likely to be tissue- and signal-

specific. A comprehensive understanding of NF-jB-dependent

gene expression must take into account the kinetics of REL- or

RELA induction and downregulation, as well as post-transcrip-

tional mechanisms that determine the duration of NF-jB

responses. Once responses to single stimuli are understood in

terms of gene expression patterns, we can begin to explore

responses to strategic combinations of stimuli, which are more

likely to represent how cells respond in vivo. Tedious though it

may be, there seems to be no way to get these insights without

conditional deletions of the Rel genes themselves. Given the

complexity of the problem, it is reasonable to consider what

benefits (other than understanding) will accrue from such

endeavors. One possibility that continues to motivate us is that

uncovering subunit-specific transcriptional mechanisms may

make it possible to selectively alter expression of small subsets

of NF-jB target genes for therapy.

Finally, it will be interesting to explore cross-talk between

the canonical and noncanonical NF-jB pathways more deeply.

One situation where this is pertinent is in the genera-

tion ⁄maintenance of mature B cells, where both pathways

have been shown to be essential. These pathways could func-

tion independently or be mutually synergistic. Our working

hypothesis is that they work synergistically, and the basis of

synergy lies in (i) genes that are activated independently by

each pathway, but function cooperatively and (ii) genes

whose transcriptional activity requires both pathways to be

activated simultaneously. A second situation where both NF-

jB pathways are likely to be pertinent is in the germinal cen-

ter reaction. Two aspects of the GC reaction make it particu-

larly interesting: (i) CD40 activates both NF-jB pathways

simultaneously and persistently, which will likely be reflected

in the transcriptional response, and (ii) BCR signaling, and

accompanying classical NF-jB-dependent gene expression,

must be incorporated into the analysis to understand the

selection process that results in affinity maturation. Indeed,

gene expression that requires interactions between the two

NF-jB pathways may be particularly sensitive to manipula-

tion. We expect that understanding cell- and stimulus-spe-

cific NF-jB transcriptional responses will be an important

aspect of future research in this area.

References

1. Dorshkind K, Montecino-Rodriguez E. Fetal

B-cell lymphopoiesis and the emergence ofB-1-cell potential. Nat Rev Immunol

2007;7:213–219.2. Abarrategui I, Krangel MS. Germline tran-

scription: a key regulator of accessibilityand recombination. Adv Exp Med Biol

2009;650:93–102.3. Hardy RR, Kincade PW, Dorshkind K.

The protean nature of cells in the B

lymphocyte lineage. Immunity2007;26:703–714.

4. Jhunjhunwala S, van Zelm MC, Peak MM,Murre C. Chromatin architecture and the

generation of antigen receptor diversity.

Cell 2009;138:435–448.5. Luning Prak ET, Monestier M, Eisenberg RA.

B-cell receptor editing in tolerance and au-toimmunity. Ann N Y Acad Sci

2011;1217:96–121.6. Nemazee D. Receptor selection in B and T

lymphocytes. Annu Rev Immunol2000;18:19–51.

7. Allman D, Pillai S. Peripheral B-cell subsets.

Curr Opin Immunol 2008;20:149–157.8. Casola S. Control of peripheral B-cell devel-

opment. Curr Opin Immunol2007;19:143–149.

9. Gerondakis S, Siebenlist U. Roles of the NF-

kappaB pathway in lymphocyte develop-ment and function. Cold Spring Harb Per-

spect Biol 2010;2:a000182.10. Vallabhapurapu S, Karin M. Regulation and

function of NF-kappaB transcription factorsin the immune system. Annu Rev Immunol

2009;27:693–733.11. Sen R, Baltimore D. Multiple nuclear factors

interact with the immunoglobulin enhancer

sequences. Cell 1986. 46: 705–716. JImmunol 2006;177:7485–7496.

12. Lenardo M, Pierce JW, Baltimore D. Pro-tein-binding sites in Ig gene enhancers



determine transcriptional activity andinducibility. Science 1987;236:1573–1577.

13. Inlay M, Alt FW, Baltimore D, Xu Y. Essen-tial roles of the kappa light chain intronic

enhancer and 3¢ enhancer in kappa rear-rangement and demethylation. Nat Immu-

nol 2002;3:463–468.14. Xu Y, Davidson L, Alt FW, Baltimore D.

Deletion of the Ig kappa light chain intronicenhancer ⁄ matrix attachment region impairs

but does not abolish V kappa J kappa rear-rangement. Immunity 1996;4:377–385.

15. Baeuerle PA, Baltimore D. A 65-kappaD sub-unit of active NF-kappaB is required for

inhibition of NF-kappaB by I kappaB. Genes

Dev 1989;3:1689–1698.16. Ghosh S, Gifford AM, Riviere LR, Tempst P,

Nolan GP, Baltimore D. Cloning of the p50DNA binding subunit of NF-kappa B:

homology to rel and dorsal. Cell1990;62:1019–1029.

17. Nolan GP, Ghosh S, Liou HC, Tempst P, Bal-timore D. DNA binding and I kappa B inhi-

bition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide. Cell

1991;64:961–969.18. Sha WC, Liou HC, Tuomanen EI, Baltimore

D. Targeted disruption of the p50 subunitof NF-kappa B leads to multifocal defects in

immune responses. Cell 1995;80:321–330.19. Beg AA, Sha WC, Bronson RT, Ghosh S, Bal-

timore D. Embryonic lethality and liverdegeneration in mice lacking the RelA com-

ponent of NF-kappa B. Nature1995;376:167–170.

20. Horwitz BH, Scott ML, Cherry SR, BronsonRT, Baltimore D. Failure of lymphopoiesis

after adoptive transfer of NF-kappaB-defi-cient fetal liver cells. Immunity

1997;6:765–772.21. Senftleben U, Li ZW, Baud V, Karin M.

IKKbeta is essential for protecting T cellsfrom TNFalpha-induced apoptosis. Immu-

nity 2001;14:217–230.22. Inlay MA, Tian H, Lin T, Xu Y. Important

roles for E protein binding sites within theimmunoglobulin kappa chain intronic

enhancer in activating Vkappa Jkappa rear-

rangement. J Exp Med 2004;200:1205–1211.

23. Das S, Nikolaidis N, Nei M. Genomic orga-nization and evolution of immunoglobulin

kappa gene enhancers and kappa deletingelement in mammals. Mol Immunol

2009;46:3171–3177.24. Fitzsimmons SP, Bernstein RM, Max EE,

Skok JA, Shapiro MA. Dynamic changes inaccessibility, nuclear positioning, recombi-

nation, and transcription at the Ig kappalocus. J Immunol 2007;179:5264–5273.

25. Goldmit M, et al. Epigenetic ontogeny ofthe Igk locus during B-cell development.

Nat Immunol 2005;6:198–203.

26. Grawunder U, Haasner D, Melchers F,Rolink A. Rearrangement and expression of

kappa light chain genes can occur withoutmu heavy chain expression during differen-

tiation of pre-B-cells. Int Immunol1993;5:1609–1618.

27. Novobrantseva TI, Martin VM, Pelanda R,Muller W, Rajewsky K, Ehlich A. Rearrange-

ment and expression of immunoglobulinlight chain genes can precede heavy chain

expression during normal B-cell develop-ment in mice. J Exp Med 1999;189:75–88.

28. Zhong H, May MJ, Jimi E, Ghosh S. Thephosphorylation status of nuclear NF-kappa

B determines its association with CBP ⁄ p300

or HDAC-1. Mol Cell 2002;9:625–636.29. Hendriks RW, Middendorp S. The pre-BCR

checkpoint as a cell-autonomous prolifera-tion switch. Trends Immunol

2004;25:249–256.30. Werner M, Hobeika E, Jumaa H. Role of

PI3K in the generation and survival of B-cells. Immunol Rev 2010;237:55–71.

31. Jimi E, et al. Activation of NF-kappaB pro-motes the transition of large, CD43+ pre-B-

cells to small, CD43) pre-B-cells. Int Immu-nol 2005;17:815–825.

32. Derudder E, et al. Development of immuno-globulin lambda-chain-positive B-cells, but

not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals. Nat

Immunol 2009;10:647–654.33. Bredemeyer AL, et al. DNA double-strand

breaks activate a multi-functional geneticprogram in developing lymphocytes. Nature

2008;456:819–823.34. Claudio E, Brown K, Siebenlist U. NF-kap-

paB guides the survival and differentiationof developing lymphocytes. Cell Death Dif-

fer 2006;13:697–701.35. Gerondakis S, et al. Unravelling the com-

plexities of the NF-kappaB signalling path-way using mouse knockout and transgenic

models. Oncogene 2006;25:6781–6799.36. Kaisho T, et al. IkappaB kinase alpha is

essential for mature B-cell development andfunction. J Exp Med 2001;193:417–426.

37. Kim S, La Motte-Mohs RN, Rudolph D, Zun-

iga-Pflucker JC, Mak TW. The role ofnuclear factor-kappaB essential modulator

(NEMO) in B-cell development and sur-vival. Proc Natl Acad Sci U S A

2003;100:1203–1208.38. Pasparakis M, Schmidt-Supprian M, Rajew-

sky K. IkappaB kinase signaling is essentialfor maintenance of mature B-cells. J Exp

Med 2002;196:743–752.39. Feng B, Cheng S, Pear WS, Liou HC. NF-kB

inhibitor blocks B-cell development at twocheckpoints. Med Immunol 2004;3:1.

40. Goudeau B, et al. IkappaBalpha ⁄ IkappaBep-silon deficiency reveals that a critical NF-

kappaB dosage is required for lymphocyte

survival. Proc Natl Acad Sci U S A2003;100:15800–15805.

41. Igarashi H, Baba Y, Nagai Y, Jimi E, GhoshS, Kincade PW. NF-kappaB is dispensable

for normal lymphocyte development inbone marrow but required for protection of

progenitors from TNFalpha. Int Immunol2006;18:653–659.

42. Johnson K, et al. Regulation of immuno-globulin light-chain recombination by the

transcription factor IRF-4 and the attenua-tion of interleukin-7 signaling. Immunity

2008;28:335–345.43. Ma S, Pathak S, Trinh L, Lu R. Interferon

regulatory factors 4 and 8 induce the

expression of Ikaros and Aiolos to down-regulate pre-B-cell receptor and promote

cell-cycle withdrawal in pre-B-cell develop-ment. Blood 2008;111:1396–1403.

44. Ma S, Turetsky A, Trinh L, Lu R. IFN regula-tory factor 4 and 8 promote Ig light chain

kappa locus activation in pre-B-cell develop-ment. J Immunol 2006;177:7898–7904.

45. Grumont RJ, Gerondakis S. Rel inducesinterferon regulatory factor 4 (IRF-4)

expression in lymphocytes: modulation ofinterferon-regulated gene expression by

rel ⁄ nuclear factor kappaB. J Exp Med2000;191:1281–1292.

46. Klug CA, et al. The v-abl tyrosine kinasenegatively regulates NF-kappa B ⁄ Rel factors

and blocks kappa gene transcription in pre-B lymphocytes. Genes Dev 1994;8:678–

687.47. Muljo SA, Schlissel MS. A small molecule

Abl kinase inhibitor induces differentiationof Abelson virus-transformed pre-B-cell

lines. Nat Immunol 2003;4:31–37.48. Scherer DC, Brockman JA, Bendall HH,

Zhang GM, Ballard DW, Oltz EM. Corepres-sion of RelA and c-rel inhibits immunoglob-

ulin kappa gene transcription andrearrangement in precursor B lymphocytes.

Immunity 1996;5:563–574.49. Guo F, Tanzer S, Busslinger M, Weih F. Lack

of nuclear factor-kappa B2 ⁄ p100 causes aRelB-dependent block in early B lympho-

poiesis. Blood 2008;112:551–559.

50. Weih F, Durham SK, Barton DS, Sha WC,Baltimore D, Bravo R. p50-NF-kappaB com-

plexes partially compensate for the absenceof RelB: severely increased pathology in

p50() ⁄ ))relB() ⁄ )) double-knockoutmice. J Exp Med 1997;185:1359–1370.

51. Alt FW, Enea V, Bothwell AL, Baltimore D.Activity of multiple light chain genes in

murine myeloma cells producing a single,functional light chain. Cell 1980;21:1–12.

52. Chen J, Alt FW. Gene rearrangement and B-cell development. Curr Opin Immunol

1993;5:194–200.53. Cadera EJ, Wan F, Amin RH, Nolla H,

Lenardo MJ, Schlissel MS. NF-kappaB



activity marks cells engaged in receptor edit-ing. J Exp Med 2009;206:1803–1816.

54. Verkoczy L, et al. A role for nuclear factorkappa B ⁄ rel transcription factors in the reg-

ulation of the recombinase activator genes.Immunity 2005;22:519–531.

55. Claudio E, Saret S, Wang H, Siebenlist U.Cell-autonomous role for NF-kappa B in

immature bone marrow B-cells. J Immunol2009;182:3406–3413.

56. Enzler T, et al. Alternative and classical NF-kappa B signaling retain autoreactive B-cells

in the splenic marginal zone and result inlupus-like disease. Immunity 2006;25:403–

415.

57. Asano J, et al. The serine ⁄ threonine kinasePim-2 is a novel anti-apoptotic mediator in

myeloma cells. Leukemia 2011;25:1182–1188.

58. Woodland RT, et al. Multiple signalingpathways promote B lymphocyte stimulator

dependent B-cell growth and survival. Blood2008;111:750–760.

59. Rowland SL, Leahy KF, Halverson R, TorresRM, Pelanda R. BAFF receptor signaling aids

the differentiation of immature B-cells intotransitional B-cells following tonic BCR sig-

naling. J Immunol 2010;185:4570–4581.60. Xue L, et al. Defective development and

function of Bcl10-deficient follicular, mar-ginal zone and B1 B-cells. Nat Immunol

2003;4:857–865.61. Enders A, Bouillet P, Puthalakath H, Xu Y,

Tarlinton DM, Strasser A. Loss of the pro-apoptotic BH3-only Bcl-2 family member

Bim inhibits BCR stimulation-induced apop-tosis and deletion of autoreactive B-cells. J

Exp Med 2003;198:1119–1126.62. Oliver PM, Vass T, Kappler J, Marrack P.

Loss of the proapoptotic protein, Bim,breaks B-cell anergy. J Exp Med

2006;203:731–741.63. Takeuchi O, Fisher J, Suh H, Harada H, Ma-

lynn BA, Korsmeyer SJ. Essential role ofBAX,BAK in B-cell homeostasis and preven-

tion of autoimmune disease. Proc Natl AcadSci U S A 2005;102:11272–11277.

64. Cancro MP, Kearney JF. B-cell positive selec-

tion: road map to the primary repertoire? JImmunol 2004;173:15–19.

65. Edry E, Melamed D. Receptor editing inpositive and negative selection of B lympho-

poiesis. J Immunol 2004;173:4265–4271.66. Grossmann M, Metcalf D, Merryfull J, Beg

A, Baltimore D, Gerondakis S. The com-bined absence of the transcription factors

Rel and RelA leads to multiple hemopoieticcell defects. Proc Natl Acad Sci U S A

1999;96:11848–11853.67. Grossmann M, O’Reilly LA, Gugasyan R,

Strasser A, Adams JM, Gerondakis S. Theanti-apoptotic activities of Rel and RelA

required during B-cell maturation involve

the regulation of Bcl-2 expression. EMBO J2000;19:6351–6360.

68. Schram BR, et al. B-cell receptor basal sig-naling regulates antigen-induced Ig light

chain rearrangements. J Immunol2008;180:4728–4741.

69. Tze LE, et al. Basal immunoglobulin signal-ing actively maintains developmental stage

in immature B-cells. PLoS Biol 2005;3:e82.70. Allman D, Lindsley RC, DeMuth W, Rudd

K, Shinton SA, Hardy RR. Resolution ofthree nonproliferative immature splenic B-

cell subsets reveals multiple selection pointsduring peripheral B-cell maturation. J

Immunol 2001;167:6834–6840.

71. Bell SE, et al. PLCgamma2 regulates Bcl-2levels and is required for survival rather than

differentiation of marginal zone and follicu-lar B-cells. Eur J Immunol 2004;34:2237–

2247.72. Cancro MP, Sah AP, Levy SL, Allman DM,

Schmidt MR, Woodland RT. xid mice revealthe interplay of homeostasis and Bruton’s

tyrosine kinase-mediated selection at multi-ple stages of B-cell development. Int Immu-

nol 2001;13:1501–1514.73. Doody GM, et al. Signal transduction

through Vav-2 participates in humoralimmune responses and B-cell maturation.

Nat Immunol 2001;2:542–547.74. Fruman DA, et al. Impaired B-cell develop-

ment and proliferation in absence of phos-phoinositide 3-kinase p85alpha. Science

1999;283:393–397.75. Jumaa H, Wollscheid B, Mitterer M, Wien-

ands J, Reth M, Nielsen PJ. Abnormal devel-opment and function of B lymphocytes in

mice deficient for the signaling adaptor pro-tein SLP-65. Immunity 1999;11:547–554.

76. Pappu R, et al. Requirement for B-cell linkerprotein (BLNK) in B-cell development. Sci-

ence 1999;286:1949–1954.77. Suzuki H, et al. Xid-like immunodeficiency

in mice with disruption of the p85alphasubunit of phosphoinositide 3-kinase. Sci-

ence 1999;283:390–392.78. Wen R, et al. Phospholipase Cgamma2 pro-

vides survival signals via Bcl2 and A1 in dif-

ferent subpopulations of B-cells. J BiolChem 2003;278:43654–43662.

79. Yamazaki T, Takeda K, Gotoh K, TakeshimaH, Akira S, Kurosaki T. Essential immuno-

regulatory role for BCAP in B-cell develop-ment and function. J Exp Med

2002;195:535–545.80. Jun JE, et al. Identifying the MAGUK pro-

tein Carma-1 as a central regulator ofhumoral immune responses and atopy by

genome-wide mouse mutagenesis. Immu-nity 2003;18:751–762.

81. Newton K, Dixit VM. Mice lacking theCARD of CARMA1 exhibit defective B lym-

phocyte development and impaired prolif-

eration of their B and T lymphocytes. CurrBiol 2003;13:1247–1251.

82. Ruefli-Brasse AA, French DM, Dixit VM.Regulation of NF-kappaB-dependent lym-

phocyte activation and development by pa-racaspase. Science 2003;302:1581–1584.

83. Ruland J, et al. Bcl10 is a positive regulatorof antigen receptor-induced activation of

NF-kappaB and neural tube closure. Cell2001;104:33–42.

84. Ruland J, Duncan GS, Wakeham A, MakTW. Differential requirement for Malt1 in T

and B-cell antigen receptor signaling.Immunity 2003;19:749–758.

85. Lin X, Wang D. The roles of CARMA1,

Bcl10, and MALT1 in antigen receptor sig-naling. Semin Immunol 2004;16:429–435.

86. Thome M, Charton JE, Pelzer C, HailfingerS. Antigen receptor signaling to NF-kappaB

via CARMA1, BCL10, and MALT1. ColdSpring Harb Perspect Biol 2010;2:a003004.

87. Andrews SF, Rawlings DJ. Transitional B-cells exhibit a B-cell receptor-specific

nuclear defect in gene transcription. JImmunol 2009;182:2868–2878.

88. Castro I, et al. B-cell receptor-mediated sus-tained c-Rel activation facilitates late transi-

tional B-cell survival through control of B-cell activating factor receptor and NF-kap-

paB2. J Immunol 2009;182:7729–7737.89. Sen R. Control of B lymphocyte apoptosis

by the transcription factor NF-kappaB.Immunity 2006;25:871–883.

90. Chung JB, Baumeister MA, Monroe JG. Cut-ting edge: differential sequestration of

plasma membrane-associated B-cell antigenreceptor in mature and immature B-cells

into glycosphingolipid-enriched domains. JImmunol 2001;166:736–740.

91. Pierce SK. Lipid rafts and B-cell activation.Nat Rev Immunol 2002;2:96–105.

92. Sproul TW, Malapati S, Kim J, Pierce SK.Cutting edge: B-cell antigen receptor signal-

ing occurs outside lipid rafts in immature B-cells. J Immunol 2000;165:6020–6023.

93. Smith SH, Cancro MP. Cutting edge: B-cellreceptor signals regulate BLyS receptor levels

in mature B-cells and their immediate pro-

genitors. J Immunol 2003;170:5820–5823.94. Hoek KL, Carlesso G, Clark ES, Khan WN.

Absence of mature peripheral B-cell popula-tions in mice with concomitant defects in B-

cell receptor and BAFF-R signaling. J Immu-nol 2009;183:5630–5643.

95. Schiemann B, et al. An essential role forBAFF in the normal development of B-cells

through a BCMA-independent pathway. Sci-ence 2001;293:2111–2114.

96. Thompson JS, et al. BAFF-R, a newly identi-fied TNF receptor that specifically interacts

with BAFF. Science 2001;293:2108–2111.97. Thompson JS, et al. BAFF binds to the

tumor necrosis factor receptor-like molecule



B-cell maturation antigen and is importantfor maintaining the peripheral B-cell popu-

lation. J Exp Med 2000;192:129–135.98. Yan M, Marsters SA, Grewal IS, Wang H,

Ashkenazi A, Dixit VM. Identification of areceptor for BLyS demonstrates a crucial role

in humoral immunity. Nat Immunol2000;1:37–41.

99. Lam KP, Kuhn R, Rajewsky K. In vivo abla-tion of surface immunoglobulin on mature

B-cells by inducible gene targeting results inrapid cell death. Cell 1997;90:1073–1083.

100. Kraus M, Alimzhanov MB, Rajewsky N,Rajewsky K. Survival of resting mature B

lymphocytes depends on BCR signaling via

the Igalpha ⁄ beta heterodimer. Cell2004;117:787–800.

101. Grande SM, Bannish G, Fuentes-Panana EM,Katz E, Monroe JG. Tonic B-cell and viral

ITAM signaling: context is everything.Immunol Rev 2007;218:214–234.

102. Srinivasan L, et al. PI3 kinase signals BCR-dependent mature B-cell survival. Cell

2009;139:573–586.103. Claudio E, Brown K, Park S, Wang H,

Siebenlist U. BAFF-induced NEMO-indepen-dent processing of NF-kappa B2 in maturing

B-cells. Nat Immunol 2002;3:958–965.104. Kayagaki N, et al. BAFF ⁄ BLyS receptor 3

binds the B-cell survival factor BAFF ligandthrough a discrete surface loop and pro-

motes processing of NF-kappaB2. Immunity2002;17:515–524.

105. Franzoso G, et al. Mice deficient in nuclearfactor (NF)-kappa B ⁄ p52 present with

defects in humoral responses, germinal cen-ter reactions, and splenic microarchitecture.

J Exp Med 1998;187:147–159.106. Weih DS, Yilmaz ZB, Weih F. Essential role

of RelB in germinal center and marginalzone formation and proper expression of

homing chemokines. J Immunol2001;167:1909–1919.

107. Opferman JT, Letai A, Beard C, SorcinelliMD, Ong CC, Korsmeyer SJ. Development

and maintenance of B and T lymphocytesrequires antiapoptotic MCL-1. Nature

2003;426:671–676.

108. Pham LV, et al. Constitutive BR3 receptorsignaling in diffuse, large B-cell lymphomas

stabilizes nuclear factor-kappaB-inducingkinase while activating both canonical and

alternative nuclear factor-kappaB pathways.Blood 2011;117:200–210.

109. Shinners NP, et al. Bruton’s tyrosine kinasemediates NF-kappa B activation and B-cell

survival by B-cell-activating factor receptorof the TNF-R family. J Immunol

2007;179:3872–3880.110. Sasaki Y, et al. Canonical NF-kappaB activ-

ity, dispensable for B-cell development,replaces BAFF-receptor signals and pro-

motes B-cell proliferation upon activation.Immunity 2006;24:729–739.

111. Basak S, Shih VF, Hoffmann A. Generationand activation of multiple dimeric transcrip-

tion factors within the NF-kappaB signalingsystem. Mol Cell Biol 2008;28:3139–3150.

112. Bren GD, Solan NJ, Miyoshi H, PenningtonKN, Pobst LJ, Paya CV. Transcription of the

RelB gene is regulated by NF-kappaB. Onco-gene 2001;20:7722–7733.

113. Lombardi L, et al. Structural and functionalcharacterization of the promoter regions of

the NFKB2 gene. Nucleic Acids Res1995;23:2328–2336.

114. Stadanlick JE, et al. Tonic B-cell antigen

receptor signals supply an NF-kappaB sub-strate for prosurvival BLyS signaling. Nat

Immunol 2008;9:1379–1387.115. Otipoby KL, et al. BAFF activates Akt and

Erk through BAFF-R in an IKK1-dependentmanner in primary mouse B-cells. Proc Natl

Acad Sci U S A 2008;105:12435–12438.116. Patke A, Mecklenbrauker I, Erdjument-Bro-

mage H, Tempst P, Tarakhovsky A. BAFFcontrols B-cell metabolic fitness through a

PKC beta- and Akt-dependent mechanism.J Exp Med 2006;203:2551–2562.

117. Grumont RJ, Strasser A, Gerondakis S. B-cellgrowth is controlled by phosphatidylinoso-

tol 3-kinase-dependent induction ofRel ⁄ NF-kappaB regulated c-myc transcrip-

tion. Mol Cell 2002;10:1283–1294.118. Kane LP, Mollenauer MN, Xu Z, Turck CW,

Weiss A. Akt-dependent phosphorylationspecifically regulates Cot induction of NF-

kappa B-dependent transcription. Mol CellBiol 2002;22:5962–5974.

119. Madrid LV, Wang CY, Guttridge DC, Scho-ttelius AJ, Baldwin AS Jr, Mayo MW. Akt

suppresses apoptosis by stimulating thetransactivation potential of the RelA ⁄ p65

subunit of NF-kappaB. Mol Cell Biol2000;20:1626–1638.

120. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR,Pfeffer LM, Donner DB. NF-kappaB activa-

tion by tumour necrosis factor requires theAkt serine-threonine kinase. Nature

1999;401:82–85.

121. Ramadani F, et al. The PI3K isoformsp110alpha and p110delta are essential for

pre-B-cell receptor signaling and B-celldevelopment. Sci Signal 2010;3:ra60.

122. Debnath I, Roundy KM, Weis JJ, Weis JH.Analysis of the regulatory role of BAFF in

controlling the expression of CD21 andCD23. Mol Immunol 2007;44:2388–2399.

123. Gorelik L, et al. Cutting edge: BAFF regu-lates CD21 ⁄ 35 and CD23 expression inde-

pendent of its B-cell survival function.J Immunol 2004;172:762–766.

124. Grumont RJ, Gerondakis S. The subunitcomposition of NF-kappa B complexes

changes during B-cell development. CellGrowth Differ 1994;5:1321–1331.

125. Liou HC, Sha WC, Scott ML, Baltimore D.Sequential induction of NF-kappa B ⁄ Rel

family proteins during B-cell terminal dif-ferentiation. Mol Cell Biol 1994;14:5349–

5359.126. Miyamoto S, Schmitt MJ, Verma IM. Quali-

tative changes in the subunit composition ofkappa B-binding complexes during murine

B-cell differentiation. Proc Natl Acad Sci U SA 1994;91:5056–5060.

127. Harhaj EW, Sun SC. Regulation of RelA sub-cellular localization by a putative nuclear

export signal and p50. Mol Cell Biol

1999;19:7088–7095.128. Huang TT, Kudo N, Yoshida M, Miyamoto

S. A nuclear export signal in the N-terminalregulatory domain of IkappaBalpha controls

cytoplasmic localization of inactive NF-kap-paB ⁄ IkappaBalpha complexes. Proc Natl

Acad Sci U S A 2000;97:1014–1019.129. Tam WF, Lee LH, Davis L, Sen R. Cytoplas-

mic sequestration of rel proteins by Ikap-paBalpha requires CRM1-dependent nuclear

export. Mol Cell Biol 2000;20:2269–2284.130. Tam WF, Wang W, Sen R. Cell-specific

association and shuttling of IkappaBalphaprovides a mechanism for nuclear NF-kap-

paB in B lymphocytes. Mol Cell Biol2001;21:4837–4846.

131. Wuerzberger-Davis SM, et al. Nuclearexport of the NF-kappaB inhibitor IkappaB-

alpha is required for proper B-cell and sec-ondary lymphoid tissue formation.

Immunity 2011;34:188–200.132. Shinohara H, Kurosaki T. Comprehending

the complex connection between PKCbeta,TAK1, and IKK in BCR signaling. Immunol

Rev 2009;232:300–318.133. Ferch U, et al. MALT1 directs B-cell recep-

tor-induced canonical nuclear factor-kappaBsignaling selectively to the c-Rel subunit.

Nat Immunol 2007;8:984–991.134. Banerjee D, Liou HC, Sen R. c-Rel-depen-

dent priming of naive T cells by inflamma-tory cytokines. Immunity 2005;23:

445–458.

135. Bajpai UD, Zhang K, Teutsch M, Sen R,Wortis HH. Bruton’s tyrosine kinase links

the B-cell receptor to nuclear factor kappaBactivation. J Exp Med 2000;191:1735–

1744.136. Petro JB, Rahman SM, Ballard DW, Khan

WN. Bruton’s tyrosine kinase is required foractivation of IkappaB kinase and nuclear fac-

tor kappaB in response to B-cell receptorengagement. J Exp Med 2000;191:1745–

1754.137. Matsuda S, et al. Critical role of class IA

PI3K for c-Rel expression in B lymphocytes.Blood 2009;113:1037–1044.



138. Park SG, et al. The kinase PDK1 integrates Tcell antigen receptor and CD28 coreceptor

signaling to induce NF-kappaB and activateT cells. Nat Immunol 2009;10:158–166.

139. Depoil D, et al. CD19 is essential for B-cellactivation by promoting B-cell receptor-

antigen microcluster formation in responseto membrane-bound ligand. Nat Immunol

2008;9:63–72.140. Tuveson DA, Carter RH, Soltoff SP, Fearon

DT. CD19 of B-cells as a surrogate kinaseinsert region to bind phosphatidylinositol

3-kinase. Science 1993;260:986–989.141. Aiba Y, Kameyama M, Yamazaki T, Tedder

TF, Kurosaki T. Regulation of B-cell devel-

opment by BCAP and CD19 through theirbinding to phosphoinositide 3-kinase.

Blood 2008;111:1497–1503.142. Okada T, Maeda A, Iwamatsu A, Gotoh K,

Kurosaki T. BCAP: the tyrosine kinase sub-strate that connects B-cell receptor to phos-

phoinositide 3-kinase activation. Immunity2000;13:817–827.

143. Damdinsuren B, et al. Single round of anti-gen receptor signaling programs naive B-

cells to receive T cell help. Immunity2010;32:355–366.

144. Sen R, Smale ST. Selectivity of the NF-{kap-pa}B response. Cold Spring Harb Perspect

Biol 2010;2:a000257.145. Arenzana-Seisdedos F, Thompson J, Rodri-

guez MS, Bachelerie F, Thomas D, Hay RT.Inducible nuclear expression of newly syn-

thesized I kappa B alpha negatively regulatesDNA-binding and transcriptional activities

of NF-kappa B. Mol Cell Biol1995;15:2689–2696.

146. Arenzana-Seisdedos F, et al. Nuclear locali-zation of I kappa B alpha promotes active

transport of NF-kappa B from the nucleus tothe cytoplasm. J Cell Sci 1997;110(Pt

3):369–378.147. Huang TT, Miyamoto S. Postrepression acti-

vation of NF-kappaB requires the amino-ter-minal nuclear export signal specific to

IkappaBalpha. Mol Cell Biol 2001;21:4737–4747.

148. Johnson C, Van Antwerp D, Hope TJ. An N-

terminal nuclear export signal is requiredfor the nucleocytoplasmic shuttling of Ikap-

paBalpha. EMBO J 1999;18:6682–6693.149. Ferrarini M, Corte G, Viale G, Durante ML,

Bargellesi A. Membrane Ig on human lym-phocytes: rate of turnover of IgD and IgM

on the surface of human tonsil cells. Eur JImmunol 1976;6:372–378.

150. Gazumyan A, Reichlin A, Nussenzweig MC.Ig beta tyrosine residues contribute to the

control of B-cell receptor signaling by regu-lating receptor internalization. J Exp Med

2006;203:1785–1794.151. Hinz M, Scheidereit C. Striking back at the

activator: how IkappaB kinase terminates

antigen receptor responses. Sci STKE2007;2007:pe19.

152. Zeng H, et al. Phosphorylation of Bcl10negatively regulates T-cell receptor-medi-

ated NF-kappaB activation. Mol Cell Biol2007;27:5235–5245.

153. Harhaj EW, Dixit VM. Deubiquitinases inthe regulation of NF-kappaB signaling. Cell

Res 2011;21:22–39.154. Chu Y, et al. B-cells lacking the tumor sup-

pressor TNFAIP3 ⁄ A20 display impaired dif-ferentiation and hyperactivation and cause

inflammation and autoimmunity in agedmice. Blood 2011;117:2227–2236.

155. Hovelmeyer N, et al. A20 deficiency in B-

cells enhances B-cell proliferation andresults in the development of autoantibod-

ies. Eur J Immunol 2011;41:595–601.156. Tavares RM, et al. The ubiquitin modifying

enzyme A20 restricts B-cell survival and pre-vents autoimmunity. Immunity

2010;33:181–191.157. Hovelmeyer N, et al. Regulation of B-cell

homeostasis and activation by the tumorsuppressor gene CYLD. J Exp Med

2007;204:2615–2627.158. Jin W, et al. Deubiquitinating enzyme CYLD

regulates the peripheral development andnaive phenotype maintenance of B-cells. J

Biol Chem 2007;282:15884–15893.159. Bystry RS, Aluvihare V, Welch KA, Kallik-

ourdis M, Betz AG. B-cells and professionalAPCs recruit regulatory T cells via CCL4. Nat

Immunol 2001;2:1126–1132.160. Reif K, et al. Balanced responsiveness to

chemoattractants from adjacent zones deter-mines B-cell position. Nature

2002;416:94–99.161. de Alboran IM, et al. Analysis of C-MYC

function in normal cells via conditionalgene-targeted mutation. Immunity

2001;14:45–55.162. Meyer-Bahlburg A, Bandaranayake AD,

Andrews SF, Rawlings DJ. Reduced c-mycexpression levels limit follicular mature B-

cell cycling in response to TLR signals. JImmunol 2009;182:4065–4075.

163. Fillatreau S, Radbruch A. IRF4 – a factor for

class switching and antibody secretion. NatImmunol 2006;7:704–706.

164. Kontgen F, et al. Mice lacking the c-relproto-oncogene exhibit defects in lympho-

cyte proliferation, humoral immunity, andinterleukin-2 expression. Genes Dev

1995;9:1965–1977.165. Owyang AM, et al. c-Rel is required for the

protection of B-cells from antigen receptor-mediated, but not Fas-mediated, apoptosis.

J Immunol 2001;167:4948–4956.166. Venkataraman L, Burakoff SJ, Sen R. FK506

inhibits antigen receptor-mediated induc-tion of c-rel in B and T lymphoid cells. J Exp

Med 1995;181:1091–1099.

167. Venkataraman L, Francis DA, Wang Z, Liu J,Rothstein TL, Sen R. Cyclosporin-A sensitive

induction of NF-AT in murine B-cells.Immunity 1994;1:189–196.

168. Hogan PG, Chen L, Nardone J, Rao A. Tran-scriptional regulation by calcium, calcineu-

rin, and NFAT. Genes Dev 2003;17:2205–2232.

169. Lynch J, et al. Calreticulin signals upstreamof calcineurin and MEF2C in a critical

Ca(2 + )-dependent signaling cascade. JCell Biol 2005;170:37–47.

170. Neilson J, Stankunas K, Crabtree GR. Moni-toring the duration of antigen-receptor

occupancy by calcineurin ⁄ glycogen-syn-

thase-kinase-3 control of NF-AT nuclearshuttling. Curr Opin Immunol

2001;13:346–350.171. Wilker PR, et al. Transcription factor Mef2c

is required for B-cell proliferation and sur-vival after antigen receptor stimulation. Nat

Immunol 2008;9:603–612.172. Grumont R, Lock P, Mollinari M, Shannon

FM, Moore A, Gerondakis S. The mitogen-induced increase in T cell size involves PKC

and NFAT activation of Rel ⁄ NF-kappaB-dependent c-myc expression. Immunity

2004;21:19–30.173. Elgueta R, Benson MJ, de Vries VC, Wasiuk

A, Guo Y, Noelle RJ. Molecular mechanismand function of CD40 ⁄ CD40L engagement

in the immune system. Immunol Rev2009;229:152–172.

174. Rickert RC, Jellusova J, Miletic AV. Signalingby the tumor necrosis factor receptor super-

family in B-cell biology and disease. Immu-nol Rev 2011;244:115–133.

175. Zarnegar B, He JQ, Oganesyan G, Hoff-mann A, Baltimore D, Cheng G. Unique

CD40-mediated biological program in B-cell activation requires both type 1 and

type 2 NF-kappaB activation pathways.Proc Natl Acad Sci U S A 2004;101:

8108–8113.176. Shih VF, Kearns JD, Basak S, Savinova

OV, Ghosh G, Hoffmann A. Kinetic con-trol of negative feedback regulators of

NF-kappaB ⁄ RelA determines their patho-

gen- and cytokine-receptor signalingspecificity. Proc Natl Acad Sci U S A

2009;106:9619–9624.177. Werner SL, Barken D, Hoffmann A.

Stimulus specificity of gene expressionprograms determined by temporal control

of IKK activity. Science 2005;309:1857–1861.

178. Pohl T, et al. The combined absence ofNF-kappa B1 and c-Rel reveals that

overlapping roles for these transcriptionfactors in the B-cell lineage are restricted

to the activation and function of maturecells. Proc Natl Acad Sci U S A

2002;99:4514–4519.



179. Jain A, et al. Specific NEMO mutationsimpair CD40-mediated c-Rel activation and

B-cell terminal differentiation. J Clin Invest2004;114:1593–1602.

180. Sen R, Baltimore D. Inducibility of kappaimmunoglobulin enhancer-binding protein

Nf-kappa B by a posttranslational mecha-nism. Cell 1986;47:921–928.

181. Gerondakis S, Grumont RJ, Banerjee A. Reg-ulating B-cell activation and survival in

response to TLR signals. Immunol Cell Biol2007;85:471–475.

182. Banerjee A, Grumont R, Gugasyan R, WhiteC, Strasser A, Gerondakis S. NF-kappaB1 and

c-Rel cooperate to promote the survival ofTLR4-activated B-cells by neutralizing Bim

via distinct mechanisms. Blood2008;112:5063–5073.



5 n fkapa b

Technology