Context-Specific BAFF-R Signaling
by the NF-kB and PI3K Pathways
Julia Jellusova,1,6Ana V. Miletic,1,6Matthew H. Cato,1Wai-Wai Lin,2Yinling Hu,4Gail A. Bishop,2,3Mark J. Shlomchik,5
and Robert C. Rickert1,*
1Program on Inflammatory Diseases, Infectious and Inflammatory Diseases Center, Sanford-Burnham Medical Research Institute,
10901 North Torrey Pines Road, La Jolla, CA 92037, USA
2Graduate Program in Immunology, The University of Iowa and the VA Medical Center, Iowa City, IA 52242, USA
3Departments of Microbiology and Internal Medicine The University of Iowa and the VA Medical Center, Iowa City, IA 52242, USA
4Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick,
MD 21701, USA
5Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
6These authors contributed equally to this work
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works
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and survival. BAFF-R signals via the noncanonical
NF-kB pathway regulated by the TRAF3/NIK/IKK1
axis. We show that deletion of Ikk1 during early B
cell development causes a partial impairment in B
cell maturation and BAFF-dependent survival, but
inactivation of Ikk1 in mature B cells does not affect
survival. We further show that BAFF-R employs
CD19 to promote survival via phosphatidylinositol
3-kinase (PI3K), and that coinactivation of Cd19
at the transitional stage. Consistent with a role for
PI3K in BAFF-R function, inactivation of PTEN medi-
ates apartialrescue ofB cell maturation and function
in Baff?/?animals. Elevated PI3K signaling also
circumvents BAFF-dependent survival in a sponta-
neous B cell lymphoma model. These findings indi-
cate that the combined activities of PI3K and IKK1
drive peripheral B cell differentiation and survival in
a context-dependent manner.
BAFF isthe mostcritical soluble factorfor peripheralBcell matu-
ration and survival, and dysregulated BAFF expression is asso-
ciated with lupus-like autoimmunity and B cell non-Hodgkin
(B-NHL)-like lymphoma (Mackay et al., 2010; Rickert et al.,
2011). BAFF-R expression is induced on newly formed B cells
that are poised to egress from the bone marrow and enter the
spleen, and is further upregulated as transitional B cells mature
to become follicular or marginal zone (MZ) B cells (Hsu et al.,
2002; Meyer-Bahlburg et al., 2008; Stadanlick et al., 2008).
Consistent with the pattern of BAFF-R expression, BAFF or
BAFF-R deficiency imposes a block at the transitional T1-T2
maturation step due to failed survival, while follicular and MZ B
cells are reduced by >90% and do not recover with age (Miller
and Hayes, 1991; Schiemann et al., 2001; Thompson et al.,
2001). Provision of a survival signal in the form of forced Bcl-2
expression rescues the transitional B cell block, leading to the
generation of follicular B cells; however, MZ B cell formation
remains impaired, indicating that BAFF-R engagement also
imparts essential differentiation signals (Rahman and Manser,
2004; Sasaki et al., 2004; Tardivel et al., 2004).
In early work distinguishing the canonical (IKK2/Nemo-depen-
dent)from the noncanonical (IKK1-dependent) NF-kB pathways,
it was observed that BAFF-R engagement efficiently induced the
cleavage of p100 (encoded by NF-kB2) into p52, allowing it to
pair with RelB to drive gene expression (Claudio et al., 2002;
Kayagaki et al., 2002; Senftleben et al., 2001). Cleavage of
p100 is enabled by IKK1-dependent phosphorylation, which
requires upstream activation by NIK (Senftleben et al., 2001;
Xiao et al., 2001). In unstimulated B cells, cytosolic TRAF3 is
bound to NIK and mediates its continual ubiquitination and
degradation (Vallabhapurapu et al., 2008; Zarnegar et al.,
2008b). BAFF-R engagement relieves this suppression by redi-
recting the ubiquitin-mediated degradation machinery to target
TRAF3, allowing for newly formed NIK to persist (Chan et al.,
(Gardam et al., 2008; Xie et al., 2007), or mice expressing a
mutated NIK molecule that cannot interact with TRAF3 (Sasaki
et al., 2008), have been found to exhibit BAFF-independent B
cell accumulation. The canonical NF-kB pathway has been
shownto prime the noncanonical pathwayby driving the expres-
sion of NF-kB2 (Dejardin et al., 2002). In this regard, studies have
shown that the B cell receptor (BCR) induces p100 to facilitate
BAFF-R signaling (Stadanlick et al., 2008). In addition, BAFF-R
has some intrinsic capacity to activate canonical NF-kB
signaling (Hildebrand et al., 2010). While inhibition of RelB by
1022 Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors
p100 is relieved by cleavage of p100 into p52, p100 has been
shown to aggregate and act as an inhibitor of p50:p65 (Basak
et al., 2007). Moreover, NIK was recently shown to be destabi-
lized by IKK1 phosphorylation (Razani et al., 2010). Thus, there
are both positive and negative feedback mechanisms regulating
the NF-kB pathways in B cells.
The majority of studies of BAFF-R signaling have focused on
signaling via the TRAF/IKK/NF-kB pathway. However, the phos-
phatidylinositol (PtdIns) 3-kinase (PI3K) pathway has also been
implicated in BAFF-R function (Baracho et al., 2011). The class
IA PI3Ks consist of three catalytic isoforms (p110a, p110b, and
p110d) that form heterodimers with adaptor subunits (p85a,
matic activity of the PI3K heterodimer. PtdIns(3,4,5)P3is also the
primary substrate for the phosphoinositide 3-phosphatase
PTEN, which directly antagonizes PI3K activity. Activation of
downstream pathways is initiated by the recruitment of effector
molecules such as PDK1, Akt, Btk, and PLCg2, which
bear pleckstrin homology (PH) domains that directly bind
PtdIns(3,4,5)P3(Baracho et al., 2011). p110d-deficient B cells
exhibit impaired BAFF-induced survival (Henley et al., 2008),
and combined inactivation of p110a/d results in failed B cell
generation or accumulation (Ramadani et al., 2010). Using Akt
phosphorylation as a surrogate readout, investigators have
observed that BAFF induces PI3K activity with both rapid and
delayed kinetics (Otipoby et al., 2008; Patke et al., 2006). Thus,
there is experimental evidence supporting a role for the PI3K
pathway in BAFF-R function, but it is unclear whether this is
a primary or ancillary role relative to the noncanonical NF-kB
Here, we report the surprising finding that acute mature B cell
survival is unaffected by the inducible loss of Ikk1, whereas early
and BAFF responsiveness. We also provide evidence that
CD19-dependent activation of the PI3K pathway is an important
contributor to BAFF-mediated B cell survival. Thus, PI3K activity
is pivotal for both BCR and BAFF-R signaling, underscoring its
significance as a therapeutic target in autoimmune disease and
B cell malignancy.
BAFF-Mediated Mature B Cell Survival Is IKK1
Although both NF-kB and PI3K pathways are activated down-
stream of BAFF-R engagement by BAFF, and loss of either
Baff or Baff-r expression results in a block at the transitional
require IKK1 and/or PI3K for maintenance and survival. To
address this issue, we generated a mouse strain in which IKK1
expression can be inducibly ablated in mature B cells by
intercrossing mice containing a loxP-flanked Ikk1 allele (Ikk1L)
(Liu et al., 2008) with the recently described hCD20TamCrestrain
(Khalil et al., 2012) bearing a loxP-regulated enhanced yellow
fluorescent protein (EYFP) reporter cassette (Srinivas et al.,
2001). Following administration of tamoxifen, Cre recombinase
is rapidly activated with concomitant expression of EYFP and
deletion of Ikk1 in B cells expressing Cre. Strikingly, we found
that deletion of Ikk1 in mature B cells did not result in depletion
of mature B cells 1 or 2 weeks following induction of Cre with
tamoxifen (Figure 1A). Flow-cytometric analysis showed that in
Ikk1L/LhCD20TamCremice, on average, over 70% of cells were
(and thus deleted Ikk1; Figure 1B). Separation of
CD21intCD23hifollicular cells and CD21hiCD23int/lowMZ B cells
7 days after tamoxifen injection showed that both subsets
of YFP+B cells persisted equally well in the spleens of
Ikk1L/LhCD20TamCremice (Figure 1B).
Consistent with our in vivo observations, in vitro survival
assays showed that B cells isolated from Ikk1L/LhCD20TamCre
mice survived as well as control B cells in media alone or with
BAFF stimulation (Figure 1C). By immunoblotting whole-cell
lysates from sorted YFP+and YFP?B cells, we confirmed that
the survival of Ikk1L/LhCD20TamCreB cells was not due to
residual expression of IKK1 protein (Figure 1D). Interestingly,
we also found that p52 was present in similar amounts in YFP+
and YFP?B cells from Ikk1L/LhCD20TamCremice, and could be
generated de novo upon BAFF stimulation (Figure 1D).
Since the hCD20TamCreinducible system does not account
for the contribution of p100 cleavage that occurred before
tamoxifen-induced Ikk1 inactivation, we intercrossed Ikk1L/L
mice with Cd19Cremice to eliminate IKK1 prior to the onset of
BAFF-R expression. Ikk1L/LCd19Cremice exhibited a 40%–
50% reduction in mature B cells (Figure 2A), but B cell develop-
ment was not blocked at the T1-T2 maturation stage as
observed in mice lacking BAFF/BAFF-R (Figures 2A and 2B)
(Sasaki et al., 2004) or mice reconstituted with Ikk1?/?fetal liver
cells (Kaisho et al., 2001). Bromodeoxyuridine (BrdU)-labeling
experiments revealed that phenotypically mature splenic B cells
in Ikk1L/LCd19Cremice exhibited a more rapid turnover, whereas
mature recirculating B cells analyzed from the bone marrow of
Ikk1L/LCd19Creand control mice had similar rates of turnover
(Figure 2C). Ikk1L/LCd19CreB cells responded to BAFF, albeit
less effectively than control B cells (Figure 2D). At the bio-
chemical level, splenic B cells from Ikk1L/LCd19Cremice showed
efficient ablation of IKK1 and impaired, but not absent, cleavage
of p100 (Figure 2E). Moreover, p100 cleavage reached comple-
tion following in vitro BAFF stimulation of Ikk1L/LCd19CreB cells
(Figure 2E). Altogether, these findings indicate that the loss of
IKK1 imposes a bottleneck at the transitional B cell stage, but
B cells that successfully traverse this stage become long-lived,
mature, recirculating B cells that do not strictly require IKK1 for
tonic BAFF-R signaling. Moreover, the results of the in vitro stim-
partially compensate for the loss of IKK1 in the processing of
p100 to generate p52.
Sustained PtdIns(3,4,5)P3Signaling Restores B Cell
Development in Baff?/?Mice
Since IKK1-dependent signaling events cannot solely account
for BAFF-R function, we sought to identify additional pathways
that may complement IKK1 activity. Several reports have shown
that BAFF-R can engage the PI3K pathway (Henley et al., 2008;
Otipoby et al., 2008; Patke et al., 2006; Woodland et al., 2008).
We confirmed these findings, showing that BAFF induced
rapid activation of Akt (Figure S1A). Addition of the p110d-
specific inhibitor IC87114 blocked Akt activation and impaired
Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors 1023
BAFF-dependent B cell survival (Figure S1B). To address the
physiologic significance of BAFF-dependent PI3K activity, we
bred PtenL/LCd19Cremice onto the BAFF-deficient background
(PtenL/LBaff?/?Cd19Cre). In PtenL/LBaff?/?Cd19CreB cells, the
absence of PTEN results in sustained activation of the PI3K
pathway due to impaired hydrolysis of the PI3K lipid product
PI(3,4,5)P3. Consistent with previous reports (Anzelon et al.,
2003; Suzuki et al., 2003), B cell-specific deletion of Pten
resulted in a skewing toward the MZ B cell fate (Figures 3A
and 3B). In contrast, Baff?/?mice exhibited a dramatic
reduction in all peripheral B cell subsets, owing to a block at
the transitional stage of maturation (Figures 3A and 3B). Strik-
ingly, in BAFF-deficient mice lacking expression of Pten, we
observed a significant recovery in B cell maturation with no
apparent bias toward the MZ B cell subset (Figures 3A and
3B). In this regard, the size of the CD21/35hiCD1d+and CD9+
B cell subsets was comparable in wild-type and PtenL/L
Baff?/?Cd19Cremice (data not shown). BCR signaling promotes
and in turn, BAFF signaling upregulates surface expression of
CD21/35 and CD23 on B cells (Gorelik et al., 2004). Here, we
found that constitutive activation of the PI3K pathway restored
CD21/35, but not CD23, expression in PtenL/LBaff?/?Cd19Cre
splenic B cells (Figure 3C; data not shown). These data indicate
that downstream of BAFF-R signaling, PI3K supports CD21/35
surface expression (Figure 3C), while CD23 expression is upre-
gulated by BAFF-R signaling in a PI3K-independent manner or
is downregulated by elevated PI3K signaling. Consistent with
flow-cytometric analyses, histological staining of spleen sec-
confirmed that PtenL/LBaff?/?Cd19Cremice did not have an
expansion of MZ B cells as was observed in PtenL/LBaff+/+
Cd19Cremice, and that the overall splenic architecture in
PtenL/LBaff?/?Cd19Cremice was similar to that in wild-type
controls (Figure S2A).
Antigen-Specific Immune Responses and Germinal
Center Formation Are Intact in PtenL/LBaff?/?Cd19Cre
Despite the paucity of mature B cells in mice lacking expression
some immunoglobulin G (IgG) is produced (Miller and Hayes,
1991; Rahman et al., 2003; Vora et al., 2003). However, the GC
response is transient, with impaired proliferation and an associ-
ated failure to form mature follicular dendritic cell networks
(Rahman and Manser, 2004; Rahman et al., 2003; Vora et al.,
Figure 1. IKK1-Deficient Mature B Cells Show Normal In Vivo Sur-
vival and BAFF-Mediated Survival In Vitro
(A) Ikk1 deletion was induced in mature B cells by tamoxifen injection of Ikk1L/L
hCD20TamCre+mice on three consecutive days. Ikk1L/LhCD20TamCre?or
Ikk1+/+hCD20TamCre+mice were used as controls (ctrl). Mice were sacrificed
1 week or 2 weeks after the last tamoxifen injection and the percentage of B
cells in the spleen was determined by flow cytometry. Graphs show means +
SD from three independent experiments.
(B) The percentage of YFP+B cells 7 days after tamoxifen injection was
comparable between CD21intCD23hifollicular B cells and CD21hiCD23int/low
MZ B cells. YFP expression was not detected in non-B cells (B220?). Data
shown are representative of two experiments.
(C)Tostudy BAFF-mediatedsurvivalinvitro,mice weresacrificedafter thelast
tamoxifen injection, and B cells were purified and stimulated with 10 ng/ml
BAFF. The percentage of viable B cells after 3 days or 5 days in culture was
determined by flow cytometry. Graphs show mean + SD from three
(D) Splenic B cells from tamoxifen-treated Ikk1L/LhCD20TamCre+mice were
stimulated overnight with 25 ng/ml BAFF or incubated in medium alone. p100
cleavage and p52 generation were visualized by western blotting. Absence of
IKK1 in Cre+cells (YFP+) was confirmed by western blot analysis. Actin was
used as loading control. Data shown are representative of two experiments.
1024 Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors
2003). As in the case of MZ B cell formation, ectopic expression
ing in the accumulation of B cells bearing an immature pheno-
type and disrupted follicular architecture (Rahman and Manser,
2004). Thus, BAFF signaling is critical for the survival of transi-
tional and mature recirculating B cells, and for promoting MZ
and GC B cell differentiation.
Although sustained PtdIns(3,4,5)P3signaling in Baff?/?mice
lacking Pten allowed for B cell development beyond the transi-
tional stage, we sought to determine whether the mature B cells
found in PtenL/LBaff?/?Cd19Cremice were functional. To this
end, we immunized PtenL/LBaff?/?Cd19Cremice and control an-
imals with nitrophenol-keyhole limpet hemocyanin (NP-KLH) in
alum and measured the relative levels of NP-specific serum
Figure 2. IKK1 Deletion Early in B Cell Development Results in an Incomplete Block in B Cell Maturation
(A) Graphs show the total cell numbers of B cells (left panel) and B cell subsets (middle and right panels) in spleens obtained from Ikk1L/LCd19Cre+and control
mice. Ikk1L/LCd19Cre?or Ikk1+/+Cd19Cre+mice were used as controls (ctrl). B cell subsets were identified by cell surface markers: B220+, total B cells;
B220+CD21intIgMlow, mature B cells; B220+CD21lowIgMhi, T1 B cells; B220+CD21hiCD23hiIgMhi, T2 B cells; B220+CD21hiCD23int/low, MZ B cells.
(B) B cell maturation in the spleen was analyzed by flow cytometry. Plots are representative of >11 mice analyzed.
(C) Mice were continuously provided BrdU in the drinking water and euthanized after 7, 14, or 21 days of treatment. Cells were harvested from the spleen and the
bone marrow and stained with a BrdU antibody and for surface markers as follows: (left) splenic follicular (B220+, IgM+, CD23hi, and CD21lo) B cells; (center) bone
marrow B cell progenitors (B220+, IgM?, and IgD?); (right) recirculating mature B cells (B220+, IgD+, and IgMlo) in the bone marrow. Four experimental and
Cd19Cre+control mice (10–15 weeks old) were used per time point and rates of turnover were calculated by linear regression analysis. Error bars represent SD.
(D) B cells from spleens enriched for mature B cells (CD23+CD43?) or from lymph nodes (LNs; B220+CD43?) were stimulated with 10 ng/ml BAFF and the
percentage of viable cells was determined by flow cytometry after 3 days and/or 5 days in culture. The graph summarizes seven samples for each genotype and
time point. Error bars represent SD.
(E) Left panel: protein lysates from freshly isolated splenic B cells were assayed for p100 cleavage by western blotting. Right panel: p100 processing to p52 in LN
B cells stimulated overnight with 25 ng/ml BAFF versus unstimulated cells.
Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors 1025
Figure 3. Constitutively Active PI3K Restores B Cell Development in Baff?/?Mice
(A) Flow cytometry of B220+ splenic cells from Pten+/+Baff+/+Cd19Cre, PtenL/LBaff+/+Cd19Cre, Pten+/+Baff?/?Cd19Cre, and PtenL/LBaff?/?Cd19Cremice. Data are
representative of eight mice per group.
(B) Absolute numbers of splenocytes and splenic B220+B cells (top panel), and splenic B cell subsets (bottom panel). Data are from five experiments with seven
mice per group; small horizontal lines indicate mean.
(C) Expression of CD21/35 on B220+-gated IgMloIgDhisplenic B cells from Pten+/+Baff+/+Cd19Cre, PtenL/LBaff+/+Cd19Cre, Pten+/+Baff?/?Cd19Cre, and PtenL/L
Baff?/?Cd19Cremice. MFI, mean fluorescence intensity.
(D) ELISA of NP-specificIgM(top)or IgG (bottom) in theseraof Pten+/+Baff+/+Cd19Cre,PtenL/LBaff+/+Cd19Cre,Pten+/+Baff?/?Cd19Cre,and PtenL/LBaff?/?Cd19Cre
mice prior to immunization (day 0), and 7 or 14 days postimmunization with 100 mg NP-KLH in alum.
(E) Flow-cytometric analysis of splenic GC B cells (B220+gated) from immunized mice (top). The graph summarizes the percentage of B220+GL7+Fas+B cells
14 days postimmunization (bottom). Error bars represent SEM.
See also Figure S2.
1026 Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors
IgM and IgG antibody at 7 and 14 days postimmunization.
PtenL/LBaff?/?Cd19Cremice produced elevated levels of NP
IgM antibody at 7 and 14 days postimmunization as compared
with Pten+/+Baff?/?Cd19Cremice, and their responses were
statistically indistinguishable from those of normal Pten+/+
Baff+/+Cd19Crecontrols (Figure 3D, top). Consistent with previ-
ously published studies (Anzelon et al., 2003; Suzuki et al.,
2003), PtenL/LBaff+/+Cd19Cremice displayed a significant reduc-
elevated PtdIns(3,4,5)P3signaling inhibits class switch recombi-
nation by terminating Foxo1-dependent Aicda transcription
(Dengler et al., 2008; Omori et al., 2006) (Figure 3D, bottom).
Correspondingly, in spite of robust antigen-specific IgM produc-
tion, the PtenL/LBaff?/?Cd19Cremice showed a virtual absence
of NP-specific IgG and resembled PtenL/LBaff+/+Cd19Cremice
in this respect (Figure 3D).
To confirm that the absence of NP-specific IgG was not due to
defective GC formation in PtenL/LBaff?/?Cd19Cremice, we
assessed the presence of GCs in immunized control mice
and PtenL/LBaff?/?Cd19Cremice. Flow-cytometric analysis of
splenocytes from immunized mice showed that unlike Pten+/+
Baff?/?Cd19Cremice, PtenL/LBaff?/?Cd19Cremice produced
abundant B220+PNA+GL7+GC B cells (Figure 3E). In fact, the
PtenL/LBaff?/?Cd19Cremice harbored a greater percentage of
GC B cells than their normal or PTEN-deficient counterparts. In
addition, staining of spleen sections with B220 and PNA showed
robust GCs in immunized PtenL/LBaff?/?Cd19Cremice, consis-
B cell responses are recovered in PtenL/LBaff?/?Cd19Cremice,
whereas repression of class switch recombination remains a
dominant effect of Pten inactivation.
PTEN-Deficient B Cells from Baff?/?Mice Are
Responsive to Extracellular Stimuli and BCR
Given the robust in vivo responses of PtenL/LBaff?/?CD19CreB
cells following immunization, we next sought to determine
whether PtenL/LBaff?/?Cd19CreB cells display the activation
and proliferative properties of mature B cells responding to spe-
cific stimuli. To this end, purified splenic Pten+/+Baff+/+CD19Cre,
Baff?/?CD19CreB cells were cultured in the presence of BAFF,
anti-igM F(ab’)2(with or withoutBAFF), agonistic CD40 antibody,
or lipopolysaccharide. Consistent with previous reports (Anzelon
et al., 2003; Suzuki et al., 2003), expression of the activation
markers CD69 and CD86 was augmented on PTEN-deficient B
cells (Figures 4A and 4B). In contrast, expression of CD69 and
CD86 was significantly reduced or absent on B cells from
BAFF-deficient animals following treatment with various stimuli
(Figures 4A and 4B). Notably, constitutive activation of the
PI3K pathway by the loss of PTEN expression in BAFF-deficient
B cells restored B cell responsiveness and induction of CD69
and CD86 expression under all conditions examined. In this re-
gard, PtenL/LBaff?/?CD19CreB cells resembled control B cells
(Figures 4A and 4B). Consistent with these data, PtenL/LBaff?/?
CD19CreB cells also proliferated robustly following stimulation
with numerous mitogenic stimuli and were comparable to
PtenL/LBaff+/+CD19CreB cells (Figure 4C). Since inhibition of
Figure 4. Upregulation of Activation Markers and Proliferation Are
Restored in Baff?/?B Cells Lacking Pten
(A) Flow-cytometric analysis of CD69 expression on Pten+/+Baff+/+Cd19Cre,
PtenL/LBaff+/+Cd19Cre, Pten+/+Baff?/?Cd19Cre, and PtenL/LBaff?/?Cd19CreB
cells following stimulation with the indicated mitogens.
(B) As in (A), expression of CD86.
(C) Purified splenic B cells from Pten+/+Baff+/+Cd19Cre, PtenL/LBaff+/+Cd19Cre,
Pten+/+Baff?/?Cd19Cre, and PtenL/LBaff?/?Cd19Cremice were stimulated as
indicated. Proliferation was determined at 48 hr by3H-thymidine incorpora-
tion. All assays were conducted in triplicate and SDs are shown as error bars.
Data are representative of three independent experiments, with two mice per
group per experiment.
(D) Pten+/+Cd19Creor PtenL/LCd19Cremature LN B cells were left untreated or
were cultured in the presence of BAFF, and cell viability was assessed by
Annexin V (AnnV) and propidium iodide (PI) staining. The graph shows the
percentage of live (AnnV?PI?) cells at each time point. Data are representative
of three experiments with five mice per group total.
See also Figure S1.
Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors 1027
PI3K impairs BAFF-R signaling (Figures S1A and S1B), we also
confirmed that sustained activation of the PI3K pathway in
PTEN-deficient B cells promotes BAFF-induced survival (Fig-
ure 4D). Thus, the competence of B cells from PtenL/LBaff?/?
CD19Cremice to respond productively to BCR engagement
and costimulation supports the strong antibody responses
PI3K-Driven B Lymphomagenesis Is Unperturbed in the
Absence of BAFF
We recently reported a model of spontaneous B cell lymphoma
in mice harboring B cell-specific deletion of genes encoding
PTEN and SHIP phosphatases (Miletic et al., 2010). This model
demonstrated not only enhanced survival of PtenL/LShipL/L
CD19Crelymphoma cells in the presence of BAFF but also a pro-
liferative response to BAFF. Moreover, PtenL/LShipL/LCD19Cre
lymphoma B cells continued to expand upon adoptive transfer
into sublethally irradiated Baff?/?recipients. Here, we sought
to determine whether BAFF is required for B lymphoma initiation
as well as progression in PtenL/LShipL/LCD19Cremice. To this
end, we crossed PtenL/LShipL/LCD19Cremice onto the Baff?/?
background (PtenL/LShipL/LBaff?/?CD19Cre). B cell development
in PtenL/LShipL/LBaff?/?CD19Cremice was comparable to that
observed in BAFF-expressing PtenL/LShipL/LCD19Cre
with B cell numbers similar to those found in wild-type controls
(Figures 5A and 5B). Strikingly, PtenL/LShipL/LBaff?/?CD19Cre
mice developed lethal lymphoma with onset and penetrance
similar to those observed in BAFF-sufficient PtenL/LShipL/L
CD19Cremice (Figure 5C). Moreover, the lymphoma cells that
expanded in PtenL/LShipL/LBaff?/?CD19Cremice were pheno-
typically similar (B220loCD5+CD11b+) to lymphoma B cells
from PtenL/LShipL/LCD19Cremice (Figure 5D). Collectively, these
data indicate that BAFF is not required for B lymphomagenesis
when PI3K signaling is highly dysregulated.
Augmented PI3K Signaling by BAFF-R Does Not Affect
the Noncanonical NF-kB Pathway and Promotes Mcl-1
To determine whether there is biochemical crosstalk or synergy
between the PI3K and NF-kB pathways downstream of BAFF-R,
we examined p100 expression
Pten+/+Cd19Creand PtenL/LCd19CreB cells. Freshly isolated B
cells from both mouse lines exhibited similar amounts of p100
and the p52 cleavage product, indicating similar in vivo re-
sponses to endogenous BAFF (Figure 6A). Accordingly, expo-
sure to BAFF in vitro resulted in efficient conversion of p100 to
deficient B cells, indicating that canonical NF-kB signaling was
not augmented by heightened activation of the PI3K pathway.
BAFF has been characterized chiefly as a prosurvival factor.
but we focused on the prosurvival Bcl-2 family member Mcl-1,
which is regulated primarily in a posttranslational manner that
requires PI3K signaling and has previously been implicated in
BAFF-R signaling (Maurer et al., 2006; Woodland et al., 2008).
Mcl-1 is phosphorylated by GSK-3b, leading to degradation of
Mcl-1. The loss of Mcl-1 is countered by Akt-mediated phos-
and p52generation in
phorylation and subsequent inactivation of GSK-3b (Maurer
et al., 2006). To examine this regulatory cascade, we measured
levels of pAkt (Ser473), pGSK-3b (Ser9), and Mcl-1 in freshly
isolated splenic B cells from Pten+/+Baff+/+Cd19Cre, PtenL/L
Baff?/?Cd19CreB cells showed elevated levels of phosphory-
lated Akt as well as phosphorylation of GSK-3b on inhibitory
serine 9, the site that is phosphorylated by Akt, as compared
with control Pten+/+Baff+/+Cd19Creor Pten+/+Baff?/?Cd19CreB
cells (Figure 6B). Consistent with these results, we also found
elevated levels of Mcl-1 in both PtenL/LBaff+/+Cd19Creand
PtenL/LBaff?/?Cd19CreB cells (Figure 6B). It is possible that
some of these differences reflect the altered distribution of B
cell subsets between these strains (Figures 3A and 3B). Never-
theless, PtenL/LBaff?/?Cd19Cremice displayed reduced total
splenic B cell numbers but a similar subset distribution
compared with normal Pten+/+Baff+/+Cd19Crecontrol mice (Fig-
ures 3A and 3B).
Mcl-1 promotes cell survival through the direct binding and
sequestration of the proapoptotic BH3 family member Bim
(Maurer et al., 2006). Correspondingly, we found an elevated
amount of Bim associated with Mcl-1 in Pten-deficient B cells
as compared with control B cells (Figure 6C). Together, these
data suggest that activation of PI3K downstream of BAFF-R
may promote B cell survival in part via maintenance of Mcl-1
expression and sequestration of Bim by Mcl-1.
BAFF-R Signaling Employs both the IKK1 and
Although it is known that PI3K is activated in B cells downstream
of BAFF-R, how PI3K is recruited to BAFF-R remains unclear.
Unlike noncanonical NF-kB signaling, which has been shown
to be dependent upon TRAF3 for activation downstream of
BAFF-R (Rickert et al., 2011), we found that Akt activation was
not affected in a positive or negative manner in mice lacking
TRAF3 in B cells (Traf3L/LCd19Cre; Figure 7A). Thus, while
TRAF3 ablation permits BAFF-independent B cell survival
(Gardam et al., 2008; Xie et al., 2007), this effect is apparently
not due to augmented PI3K signaling.
Downstream of the BCR, PI3K p110d can act on membrane
substrates via p85a-mediated recruitment to the transmem-
brane adaptor CD19 as well as to the cytosolic adaptor BCAP
(Baracho et al., 2011; So and Fruman, 2012). To determine
whether CD19 may also act as a coreceptor for BAFF-R
signaling, we treated Cd19+/+and Cd19?/?(aka Cd19Cre/Cre)
splenic and lymph node (data not shown) B cells with BAFF
and examined them for phosphorylation of CD19 on the p85-
binding sites Y513 and Akt S473. We found that BAFF-R binding
sion of CD19 augmented Akt activation (Figure 7B). Impaired
BAFF-R signaling correlated with reduced survival of Cd19?/?
B cells cultured in the presence of BAFF (Figure 7C). Together,
these results indicate that CD19 is a critical component of
BAFF-R signaling that may recruit PI3K to BAFF-R in a manner
analogous to its role in BCR signaling.
In agreement with earlier findings, Cd19?/?mice displayed a
modest reduction in mature B cells and a near absence of MZ
1028 Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors
B cells (Figure 7D). However, unlike Baff?/?mice, the T2 popula-
tionwas unaffected(Figures 3A,3B,and 7D,top).Thus, to deter-
mine whether BAFF-R may differentially utilize the IKK1 and
CD19/PI3K pathways in transitional, mature, and MZ B cell sub-
sets, we generated mice lacking both CD19 and IKK1 in B cells
(Ikk1L/LCd19Cre/Cre). Strikingly, these mice exhibited a strong
block in peripheral B cell maturation that was comparable to
that observed in Baff?/?mice (Figures 3A, 3B, and 7D, bottom).
Indeed, Ikk1L/LCd19Cre/CreB cells were nonresponsive to BAFF
stimulation in vitro (Figure 7C). These findings suggest that the
Figure 5. Lymphoma Development in PtenL/LShipL/LCd19CreMice Occurs in a BAFF-Independent Manner
(A) Flow-cytometric analysisof B220+-gated splenic cells from Pten+/+Ship+/+Baff+/+Cd19Cre,Pten+/+Ship+/+Baff?/?Cd19Cre,PtenL/LShipL/LBaff+/+Cd19Cre,and
PtenL/LShipL/LBaff?/?Cd19Cremice. Data are representative of two independent experiments with at least two mice per group.
(B) Absolute numbers of splenocytes and splenic B cells (n = 3 mice per group; small horizontal lines indicate mean).
(C) Kaplan-Meier survival curve of Pten+/+Ship+/+Baff+/+Cd19Cre(n = 7), PtenL/LShipL/LBaff+/+Cd19Cre(n = 6), and PtenL/LShipL/LBaff?/?Cd19Cre(n = 9) mice.
(D)Expansion ofB220?/lowCD19+lymphomaBcellsinperipheralbloodofPten+/+Ship+/+Baff+/+Cd19Cre,PtenL/LShipL/LBaff+/+Cd19Cre,and PtenL/LShipL/LBaff?/?
Cd19Cremice as determined by flow cytometry at the indicated time points. Data shown are from two representative animals for each group. The PtenL/LShipL/L
Baff+/+Cd19Creanimal shown in the bottom row died before 9 months of age.
Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors 1029
signaling in newly formed B cells.
It has been shown in numerous studies that BAFF depletion
causes the rapid loss of transitional, mature, and GC B cells.
to occur similarly in these B cell subsets. Early studies showed
that fetal-liver-derived B cells from Ikk1?/?mice presented a
block at the late transitional (T2) B cell stage (Kaisho et al.,
2001), but we found that Ikk1 inactivation in early B cells resulted
in only a partial block at the T2 stage. This apparent discrepancy
might be explained by the recent discovery of a role for IKK1 in
early B cell generation, and perhaps a greater dependence on
IKK1 activity for fetal- versus bone-marrow-derived B cells (Bal-
khi et al., 2012). BrdU-labeling studies revealed that splenic B
cells bearing a mature phenotype exhibited a higher turnover in
Ikk1L/LCd19Cremice, suggesting that they were relatively short-
lived. However, turnover of mature recirculating B cells in the
bone marrow of Ikk1L/LCd19Cremice was unaffected by the
loss of IKK1, consistent with results obtained from the inducible
nation for these findings is that inhibitory p100 accumulates in
transitional B cells and descendant mature B cells in the spleens
of Ikk1L/LCd19Cremice, predisposing them to apoptosis and
failed entry into the long-lived mature recirculating B cell pool.
with our previous observation of intact B cell maturation and sur-
vival in knockin mice expressing a mutant IKK1 molecule that
cannot be phosphorylated by NIK (IKKAA) (Mills et al., 2007). In
contrast, the IKKAAmice exhibit a complete block in GC B cell
Figure 6. Augmented PI3K Signaling by
BAFF-R Does Not Affect the Noncanonical
NF-kB Pathway and
Cd19CreB cells were left untreated or were
cultured in the presence of BAFF or anti-igM
F(ab0)2fragments. Activation of noncanonical NF-
kB was determined by western blotting with anti-
bodies against p100/p52. Membranes were strip-
ped and reprobed for actin as a loading control.
(B) Western blots of protein lysates from freshly
Cd19Cre, Pten+/+Baff?/?Cd19Cre, or PtenL/LBaff?/?
Cd19Cresplenic B cells probed with pAkt1 (S473),
GSK-3b (S9), Mcl-1, or Akt1 antibodies.
(C) Pten+/+Cd19Creand PtenL/LCd19CreB cells
were left untreated or were treated with BAFF.
Lysates were generated and Mcl-1 was immuno-
precipitated. Immunoprecipitates were resolved
by SDS-PAGE and membranes were probed with
antibodies against Bim and Mcl-1.
differentiation (Mills etal.,2007), suggest-
ing that BAFF-R/NIK/IKK1 signaling may
be important for priming the survival and
differentiation pathways that are set in
place after antigen encounter. Expression of a constitutively
active form of IKK2 or disruption of NIK degradation also allows
for BAFF-independent B cell maturation (Sasaki et al., 2006,
2008). Elevated NIK activity has been shown to activate the
canonical NF-kB pathway as well as the noncanonical pathway
(Zarnegar et al., 2008a). Thus, the B cell phenotypes observed
in mice expressing constitutively active IKK2 or NIK may have
similar biochemical underpinnings in abnormally augmenting
canonical NF-kB-dependent gene transcription.
Given that inactivation of the noncanonical NF-kB pathway is
insufficient to explain the biologic effects of BAFF depletion on
mature B cells, we focused on the PI3K pathway, which has pre-
viously been implicated in BAFF-R function (Baracho et al.,
proliferation, survival, and differentiation. Correspondingly,
BAFF stimulation also primes B cells for cell-cycle entry and
protein synthesis (Huang et al., 2004; Patke et al., 2006). These
effector pathways likely account, in part, for the observed
defects in the MZ and GC B cell compartments in mice bearing
defects in PI3K/Akt signaling (Calamito et al., 2010; Clayton
et al., 2002; Zhang et al., 2012).
Themajority ofstudiesofPI3K functioninBcellshavefocused
onBCR-induced PI3Kactivity,including the recruitment ofCD19
as a coreceptor. In this regard, inactivation of CD19 or p110d
(Clayton et al., 2002; Engel et al., 1995; Okkenhaug et al., 2002;
Rickert et al., 1995); however, dual ablation of p110a/d leads to
a nearly complete block in B cell development at the pro-B cell
stage (Ramadani et al., 2010). Here, we show that CD19 contrib-
utes to BAFF-mediated survival, consistent with BAFF-induced
CD19 phosphorylation and Akt activation. Intriguingly, this
finding suggests that BAFF-R employs signaling components
1030 Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors
associated with the BCR in a ‘‘coreceptor’’ capacity. This asser-
tion is supported by the recent work of Schweighoffer et al.
(2013), who reported a role for Syk in BAFF-R signaling. Findings
that BAFF activates Btk also support the possible linkage of the
Figure 7. BAFF-Induced Signaling Is Atten-
uated in B Cells Lacking ExpressionofCD19
(A) Western blots of protein lysates from Traf3+/+
Cd19Creor Traf3L/LCd19Cresplenic B cells treated
for the indicated time points with BAFF were pro-
bed with pAkt1 (S473) or tAkt1 antibodies.
(B) Western blots of protein lysates from Cd19+/+
or Cd19?/?splenic B cells treated for the indicated
time points with 25 ng/ml BAFF were probed with
pCD19 (Y513), CD19, pAkt1 (S473), or tAkt1
(C) Cd19+/+or Cd19?/?LN B cells were left un-
treated or were cultured in the presence of
25 ng/ml BAFF and the percentage of viable cells
was determined by flow cytometry after 3 days
and/or 5 days in culture (left panel). Graphs sum-
marize data from three individual mice in technical
triplicates per genotype. LN B cells from Ikk1L/L
Cd19Cre/Cre(IKK1 and CD19 double-deficient) and
control mice were treated with 10 ng/ml BAFF or
were cultured in medium alone, and cell viability
was assessed 3 days later (right panel). Graphs
summarize results from three independent exper-
iments with seven control samples and three
Ikk1L/LCd19Cre/Cresamples in total. These mea-
surements were part of the experiments described
Ikk1L/LCd19Cre/Cresamples can be directly com-
pared with the Ikk1L/LCd19Cre/+samples shown in
Figure 2C. Error bars represent SD.
(D) Total splenic B cell numbers and cell numbers
of the indicated B cell subsets from Cd19?/?and
control mice are shown in the top panel. B cell
subsets were defined as in Figure 2A. Analysis of
total cell numbers for Ikk1L/LCd19Cre/Creand con-
trol mice is shown in the bottom panel. These mice
were analyzed in parallel with mice presented in
Figure 2A; therefore, results shown for Ikk1L/L
Cd19Cre/Cremice can be directly compared with
data from the Ikk1L/LCd19Cre/+mice shown in
ners et al., 2007). Thus, previous studies
showing that the BCR is required for
continued B cell survival may have
incorporated homeostatic signaling by
BAFF-R (Lam et al., 1997).
To further evaluate the PI3K pathway in
BAFF-R signaling, we performed gain-of-
function studies by inactivating Pten in B
cells. This alteration is similar to that
achieved by expressing constitutively
active PI3K (p110), in that PTEN loss
leads to the sustained presence of
PtdIns(3,4,5)P3. We previously showed
that PTEN loss leads to the preferential
expansion of the MZ and B-1 B cell compartments, and comple-
loss of PTEN supports B cell maturation and function in BAFF-
deficient mice. Interestingly, the distribution of peripheral B cell
subsets in PtenL/LBaff?/?Cd19Cremice is more similar to that
Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors 1031
observed inwild-typeanimalsthanto thatfoundinPtenL/LBaff+/+
B cell defects in Baff?/?mice. Moreover, unlike ectopic Bcl-2
expression (Rahman and Manser, 2004; Tardivel et al., 2004),
the partialrescueofthe BAFFdefect isnotconfinedtoenhanced
B cell survival, but also extends to B cell differentiation and anti-
gen-dependent responses. That said, a full restoration of the
mature recirculating B cell pool is not observed in PtenL/LBaff?/?
Cd19Cremice, likely reflecting the importance of IKK1 activity at
the transitional B cell stage. This hypothesis is further supported
by the phenotype of Cd19Cre/CreIkk1L/Ldouble-deficient mice,
underscoring a synergistic relationship between CD19/PI3K
and IKK1 signaling.
BAFF induces the transcription of the prosurvival factors A1,
Bcl-xL, and Pim2 (Enzler et al., 2006; Hatada et al., 2003; Hsu
et al., 2002). Consistent with the role of BAFF in generating T2
B cells, early studies of Bcl-xL?/?mice revealed a reduced per-
centage of IgM+IgD?B cells (Motoyama et al., 1995). However,
the B cells that overcome this bottleneck exhibit normal survival
as mature recirculating cells (Motoyama et al., 1995), which may
be similar to the phenotype we observed in Ikk1L/LCd19Cremice.
Although Pim2?/?and NF-kB2?/?B cellsshowed similardefects
in BAFF-mediated survival in vitro (Enzler et al., 2006), inactiva-
tion of all three Pim genes resulted in only a subtle defect in
peripheral B cells in younger mice (Mikkers et al., 2004). Induc-
tion of A1 transcription by BAFF is not strictly correlated with
increased protein expression (Hatada et al., 2003). Moreover,
A1 represents a quartet of highly similar genes, one of which
(A1a)has beenshownto bedispensable forBAFF-mediated sur-
vival, suggesting that A1 induction by BAFF may not be critical
(Hatada et al., 2003).
Mcl-1 has been linked to BAFF signaling (Giltiay et al., 2010;
Woodland et al., 2008), but it is not a transcriptional target of
NF-kB. Mcl-1 protein is extremely labile and earlier studies
have shown that it is essential for early B cell generation (Opfer-
man et al., 2003). More recently, Vikstrom et al. (2010) demon-
strated that Mcl-1 is essential for GC and, to a lesser extent,
follicular B cell survival. By contrast, loss of Bcl-xL is inconse-
quential for GC B cell differentiation and survival (Vikstrom
et al., 2010). We show that PTEN loss promotes Mcl-1 expres-
sion, likely due to inactivation of GSK-3 by Akt and resultant
disruption of GSK-3-dependent Mcl-1 degradation (Maurer
et al., 2006). Thus, our data suggest that Mcl-1 regulation is an
important target of PI3K-mediated survival in mature B cells.
Inhibition of the PI3K pathway is of broad interest for applica-
tions in oncology, including the treatment of B cell malignancies.
The first-in-class small-molecule inhibitor GS-1101, which is
selective for p110d, has met with considerable success in the
clinic and is now entering phase 3 clinical trials for the treatment
of B cell chronic lymphocytic leukemia. In addition, phase 2 trials
are under way for the use of GS-1101 in the treatment of indolent
B-NHL (follicular lymphoma, small lymphocytic lymphoma,
lymphoplasmacytoid lymphoma, and MZ lymphoma). The
efficacy of these inhibitors is largely attributed to the inhibition
of BCR-mediated signaling. However, our findings suggest a
reappraisal of the molecular basis of theseBCR-targeting strate-
gies to take into account the consequences of impaired BAFF-R
signaling that may nonetheless be acting through the BCR com-
plex. As such, BAFF-depletion regimens may be effective in
combined therapies with small-molecule inhibitors targeting
BCR signaling. Based upon the mouse lymphoma studies
presented here, we would also predict that BAFF-depletion
therapy would not be effective in lymphoma cases where PI3K
signaling is elevated.
hCD20TamCreanimals (Khalil etal.,2012)wereintercrossedwithmice carrying
the rosa26-flox-STOP-YFP allele (Srinivas et al., 2001), in which YFP is
expressed upon Cre activation. Ikk1L/LCD20TamCreand control animals
were injected i.p. with 1 mg tamoxifen (Sigma-Aldrich) + 10% ethanol in olive
oil on three subsequent days. PtenL/LCd19Cremice (Anzelon et al., 2003) were
crossed to Baff?/?(Schiemann et al., 2001) mice to generate a mouse line with
B cell-specific deletion of Pten and absence of Baff expression in all tissues
(PtenL/LBaff?/?Cd19Cre). Ikk1L/Land Cd19Cremouse lines were intercrossed
to obtain IKK1-deficient mice (Ikk1L/LCd19Cre) and IKK1 and CD19 double-
deficient mice (Ikk1L/LCd19Cre/Cre). All animals were maintained in the animal
facility of the Sanford-Burnham Medical Research Institute (SBMRI). All proto-
cols were approved by the Institutional Animal Care and Use Committee at
SBMRI and were carried out in accordance with institutional guidelines and
Spleens were embedded in Tissue-Tek O.C.T. (Sakura Finetek) and frozen
at ?80?C. Acetone-fixed sections were blocked for 1 hr with 1% BSA + 5%
fetal bovine serum (FBS) in PBS and stained with a combination of various
antibodies (Moma-1-bio, CD3-APC, B220-PE, B220-FITC, and PNA-FITC)
for 2 hr at room temperature or overnight at 4?C, and streptavidin-Cy3 was
added in a second staining step. Images were acquired on a Zeiss Axio
ImagerM1 microscope (Zeiss).
Flow Cytometry and Antibodies
Single-cell suspensions were prepared, counted, and stained with antibodies
according to standard procedures. The following antibody clones were
obtained from eBioscience: CD3 (145-2C11), IgM (II/41), IgD (11-26), CD19
(ID3), B220 (RA3-6B2), CD11b (M1/70), CD43 (S7), CD21 (4E3), CD23
(B3B4), CD4 (GK1.5), and CD8 (53-6.7). Biotinylated reagents were detected
with streptavidin conjugated to a fluorescent marker (BD Biosciences). All
data were collected on a FACSCanto flow cytometer (BD Biosciences).
Immunizations and ELISA
Mice were immunized i.p. with 100 mg NP-KLH precipitated in alum (Imject;
Pierce), and serum was collected 0, 7, and 14 days postimmunization. Costar
EIA/RIA plates (Corning) were coated with 10 mg/ml NP23-BSA (Biosearch
Technologies) in PBS containing 0.05% sodium azide. Following blocking
with 0.25% BSA in PBS, serial dilutions of the indicated serum samples
were added. Alkaline phosphatase-labeled anti-mouse IgM or IgG antibody
(Southern Biotech) and p-nitrophenylphosphate substrate (Sigma-Aldrich)
were used for colorimetric detection at 405 nm using an ELx808 plate reader
with KC4 software (BioTek Instruments).
Cell Culture, Survival, and Proliferation Assays
B cell purification and in vitro stimulation were performed as previously
described (Miletic et al., 2010). For survival assays, purified splenic or lymph
node B cells were plated at a concentration of 1 3 106cells/ml in 10% media.
scatter properties of the cells or by using the AnnV-FITC Apoptosis Detection
Kit (BioVision) according to the manufacturer’s instructions. For inhibition of
PI3K p110d, cells were pretreated with 10 mM IC87114 in DMSO (ICOS).
Immunoblotting and Immunoprecipitations
Purified B cells were stimulated with 1 mg/ml anti-igM F(ab0)2or with 25 ng/ml
BAFF for the indicated time points, and then lysed on ice with RIPA buffer
1032 Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors
(PBS, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 10 mM EDTA) supplemented
with a protease inhibitor cocktail (Boehringer Mannheim), 10 mM sodium fluo-
ride, and 1 mM Na3VO4, and phenylmethanesulfonylfluoride. Equal protein
amounts were resolved on 10% Bis-Tris gels (Bio-Rad or Invitrogen) followed
by western blotting for the indicated proteins. Antibodies raised against total
IKK1, phospho-Akt (S473), total Akt, p100/p52, phospho-CD19 (Y513), total
CD19, phospho-GSK-3b (S9), actin, and Bim were obtained from Cell
Signaling Technology. Anti-Mcl-1 was purchased from Rockland Immu-
nochemicals. Primary antibodies were detected using horseradish peroxi-
dase-labeled donkey anti-rabbit (Jackson Immunoresearch) or anti-mouse
For coimmunoprecipitation, B cells were lysed in lysis buffer for 20 min on
ice. Clarified lysates were incubated with 2 mg anti-Mcl-1 or control IgG anti-
bodies overnight at 4?C. Protein A/G beads (GE Healthcare) were added for
1hrat 4?C. Immunoprecipitateswerewashed asdescribed previously (Maurer
et al., 2006) and western blotting was performed as described above.
Mice were provided 0.5 mg/ml BrdU (Sigma) + 2% sucrose in drinking water
for up to 21 days. Bone marrow and splenic cells were isolated on days 7,
14, and 21, and stained with antibodies as indicated. After surface staining,
the cells were fixed with BD Cytofix/Cytoperm (BD Biosciences) and permea-
bilized with permeabilization buffer (eBioscience), followed by permeabiliza-
tion with 0.1% Triton X-100 (Sigma), a second fixation, and DNase (Sigma)
treatment. The cells were then stained with an BrdU antibody (Invitrogen).
Software and Statistical Analysis
Gimp (GNU Image Manipulation Program) and GraphPad Prism (GraphPad
Software) were used for image editing and statistical evaluation, respectively.
Thesignificance of observed differenceswasevaluated by unpaired t test. The
obtained p values are indicated as follows: ***p < 0.001, **p < 0.005, *p < 0.05.
Supplemental Information includes two figures and can be found with this
article online at http://dx.doi.org/10.1016/j.celrep.2013.10.022.
We thank the members of the Rickert laboratory for discussions, and
Dr. D. Nemazee (TSRI, La Jolla, CA) for facilitating the transfer of the Baff?/?
mice. This work was supported by NIH grants AI041649, HL088686, and
RR026280 (to R.C.R); AI043603 and AR44077 (to M.J.S.); and AI49993 (to
G.A.B). G.A.B. received resources and use of facilities from the Iowa City
VAMC. A.V.M. was supported by NIH F32 fellowship CA132350. J.J. was
supported by fellowships from the Deutsche Forschungsgemeinschaft and
the Arthritis National Research Foundation.
Received: March 20, 2013
Revised: August 10, 2013
Accepted: October 10, 2013
Published: November 14, 2013
Anzelon, A.N., Wu, H., and Rickert, R.C. (2003). Pten inactivation alters
peripheral B lymphocyte fate and reconstitutes CD19 function. Nat. Immunol.
Balkhi, M.Y., Willette-Brown, J., Zhu, F., Chen, Z., Liu, S., Guttridge, D.C.,
Karin, M., and Hu, Y. (2012). IKKa-mediated signaling circuitry regulates early
B lymphopoiesis during hematopoiesis. Blood 119, 5467–5477.
Baracho, G.V., Miletic, A.V., Omori, S.A., Cato, M.H., and Rickert, R.C. (2011).
Emergence of the PI3-kinase pathway as a central modulator of normal and
aberrant B cell differentiation. Curr. Opin. Immunol. 23, 178–183.
Basak, S., Kim, H., Kearns, J.D., Tergaonkar, V., O’Dea, E., Werner, S.L.,
Benedict, C.A., Ware, C.F., Ghosh, G., Verma, I.M., and Hoffmann, A.
(2007). A fourth IkappaB protein within the NF-kappaB signaling module.
Cell 128, 369–381.
Calamito, M., Juntilla, M.M., Thomas, M., Northrup, D.L., Rathmell, J.,
Birnbaum, M.J., Koretzky, G., and Allman, D. (2010). Akt1 and Akt2 promote
peripheral B-cell maturation and survival. Blood 115, 4043–4050.
Chan, T.D., Gardam, S., Gatto, D., Turner, V.M., Silke, J., and Brink, R. (2010).
In vivo control of B-cell survival and antigen-specific B-cell responses.
Immunol. Rev. 237, 90–103.
Claudio, E., Brown, K., Park, S., Wang, H., and Siebenlist, U. (2002). BAFF-
induced NEMO-independent processing of NF-kappa B2 in maturing B cells.
Nat. Immunol. 3, 958–965.
Clayton, E., Bardi, G., Bell, S.E., Chantry, D., Downes, C.P., Gray, A.,
Humphries, L.A., Rawlings, D., Reynolds, H., Vigorito, E., and Turner, M.
(2002). A crucialrole for the p110delta subunit of phosphatidylinositol 3-kinase
in B cell development and activation. J. Exp. Med. 196, 753–763.
Dejardin, E., Droin, N.M., Delhase, M., Haas, E., Cao, Y., Makris, C., Li, Z.W.,
Karin, M., Ware, C.F., and Green, D.R. (2002). The lymphotoxin-beta receptor
induces different patterns of gene expression via two NF-kappaB pathways.
Immunity 17, 525–535.
Dengler, H.S., Baracho, G.V., Omori, S.A., Bruckner, S., Arden, K.C.,
Castrillon, D.H., DePinho, R.A., and Rickert, R.C. (2008). Distinct functions
for the transcription factor Foxo1 at various stages of B cell differentiation.
Nat. Immunol. 9, 1388–1398.
Engel, P., Zhou, L.J., Ord, D.C., Sato, S., Koller, B., and Tedder, T.F. (1995).
Abnormal B lymphocyte development, activation, and differentiation in mice
that lack or overexpress the CD19 signal transduction molecule. Immunity 3,
Enzler, T., Bonizzi, G., Silverman, G.J., Otero, D.C., Widhopf, G.F., Anzelon-
Mills, A., Rickert, R.C., and Karin, M. (2006). Alternative and classical
NF-kappa B signaling retain autoreactive B cells in the splenic marginal zone
and result in lupus-like disease. Immunity 25, 403–415.
Gardam,S.,Sierro,F.,Basten, A.,Mackay,F.,and Brink, R.(2008).TRAF2and
TRAF3 signal adapters act cooperatively to control the maturation and survival
signals delivered to B cells by the BAFF receptor. Immunity 28, 391–401.
Giltiay, N.V., Lu, Y., Allman, D., Jørgensen, T.N., and Li, X. (2010). The adaptor
molecule Act1 regulates BAFF responsiveness and self-reactive B cell
selection during transitional B cell maturation. J. Immunol. 185, 99–109.
Gorelik, L., Cutler, A.H., Thill, G., Miklasz, S.D., Shea, D.E., Ambrose, C.,
Bixler, S.A., Su, L., Scott, M.L., and Kalled, S.L. (2004). Cutting edge: BAFF
regulates CD21/35 and CD23 expression independent of its B cell survival
function. J. Immunol. 172, 762–766.
Hatada, E.N., Do, R.K.G., Orlofsky, A., Liou, H.-C., Prystowsky, M.,
MacLennan, I.C.M., Caamano, J., and Chen-Kiang, S. (2003). NF-kappa B1
p50 is required for BLyS attenuation of apoptosis but dispensable for pro-
cessing of NF-kappa B2 p100 to p52 in quiescent mature B cells.
J. Immunol. 171, 761–768.
Henley, T., Kovesdi, D., and Turner, M. (2008). B-cell responses to B-cell
activation factor of the TNF family (BAFF) are impaired in the absence of
PI3K delta. Eur. J. Immunol. 38, 3543–3548.
Hildebrand, J.M., Luo, Z., Manske, M.K., Price-Troska, T., Ziesmer, S.C., Lin,
W., Hostager, B.S., Slager, S.L., Witzig, T.E., Ansell, S.M., et al. (2010). A
BAFF-R mutation associated with non-Hodgkin lymphoma alters TRAF
recruitment and reveals new insights into BAFF-R signaling. J. Exp. Med.
Hsu, B.L., Harless, S.M., Lindsley, R.C., Hilbert, D.M., and Cancro, M.P.
(2002). Cutting edge: BLyS enables survival of transitional and mature B cells
through distinct mediators. J. Immunol. 168, 5993–5996.
Huang, X., Di Liberto, M., Cunningham, A.F., Kang, L., Cheng, S., Ely, S., Liou,
H.C., Maclennan,I.C.,and Chen-Kiang, S.(2004).Homeostaticcell-cyclecon-
trol by BLyS: Induction of cell-cycle entry but not G1/S transition in opposition
to p18INK4c and p27Kip1. Proc. Natl. Acad. Sci. USA 101, 17789–17794.
Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors 1033
Kaisho, T., Takeda, K., Tsujimura, T., Kawai, T., Nomura, F., Terada, N., and
Akira, S. (2001). IkappaB kinase alpha is essential for mature B cell develop-
ment and function. J. Exp. Med. 193, 417–426.
Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, D.M.,
Grewal, I.S., Cochran, A.G., Gordon, N.C., Yin, J., et al. (2002). BAFF/BLyS
receptor 3 binds the B cell survival factor BAFF ligand through a discrete
Khalil, A.M., Cambier, J.C., and Shlomchik, M.J. (2012). B cell receptor signal
transduction in theGCis short-circuitedby high phosphatase activity.Science
Lam, K.P., Ku ¨hn, R., and Rajewsky, K. (1997). In vivo ablation of surface
immunoglobulin on mature B cells by inducible gene targeting results in rapid
cell death. Cell 90, 1073–1083.
Liu, B., Xia, X., Zhu, F., Park, E., Carbajal, S., Kiguchi, K., DiGiovanni, J.,
Fischer, S.M., and Hu, Y. (2008). IKKalpha is required to maintain skin homeo-
stasis and prevent skin cancer. Cancer Cell 14, 212–225.
Mackay, F., Figgett, W.A., Saulep, D., Lepage, M., and Hibbs, M.L. (2010).
B-cell stage and context-dependent requirements for survival signals from
BAFF and the B-cell receptor. Immunol. Rev. 237, 205–225.
Maurer, U., Charvet, C., Wagman, A.S., Dejardin, E., and Green, D.R. (2006).
Glycogen synthase kinase-3 regulates mitochondrial outer membrane perme-
abilization and apoptosis by destabilization of MCL-1. Mol. Cell 21, 749–760.
Meyer-Bahlburg, A.,Andrews, S.F., Yu, K.O., Porcelli,S.A., and Rawlings,D.J.
(2008). Characterization of a late transitional B cell population highly sensitive
to BAFF-mediated homeostatic proliferation. J. Exp. Med. 205, 155–168.
Berns, A. (2004). Mice deficient for all PIM kinases display reduced body size
and impaired responses to hematopoietic growth factors. Mol. Cell. Biol. 24,
Miletic, A.V., Anzelon-Mills, A.N., Mills, D.M., Omori, S.A., Pedersen, I.M.,
Shin, D.M., Ravetch, J.V., Bolland, S., Morse, H.C., 3rd, and Rickert, R.C.
(2010). Coordinate suppression of B cell lymphoma by PTEN and SHIP
phosphatases. J. Exp. Med. 207, 2407–2420.
Miller, D.J., and Hayes, C.E. (1991). Phenotypic and genetic characterization
of a unique B lymphocyte deficiency in strain A/WySnJ mice. Eur. J. Immunol.
Mills,D.M., Bonizzi, G., Karin, M., and Rickert, R.C. (2007).Regulationof late B
cell differentiation by intrinsic IKKalpha-dependent signals. Proc. Natl. Acad.
Sci. USA 104, 6359–6364.
Motoyama, N., Wang, F., Roth, K.A., Sawa, H., Nakayama, K., Nakayama, K.,
Negishi, I., Senju, S., Zhang, Q., Fujii, S., et al. (1995). Massive cell death of
immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science
Okkenhaug, K., Bilancio, A., Farjot, G., Priddle, H., Sancho, S., Peskett, E.,
Pearce, W., Meek, S.E., Salpekar, A., Waterfield, M.D., et al. (2002). Impaired
B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice.
Science 297, 1031–1034.
Omori, S.A., Cato, M.H., Anzelon-Mills, A., Puri, K.D., Shapiro-Shelef, M.,
Calame, K., and Rickert, R.C. (2006). Regulation of class-switch recombina-
tion and plasma cell differentiation by phosphatidylinositol 3-kinase signaling.
Immunity 25, 545–557.
Opferman, J.T., Letai, A., Beard, C., Sorcinelli, M.D., Ong, C.C., and Kors-
meyer, S.J. (2003). Development and maintenance of B and T lymphocytes
requires antiapoptotic MCL-1. Nature 426, 671–676.
Otipoby, K.L., Sasaki, Y., Schmidt-Supprian, M., Patke, A., Gareus, R.,
Pasparakis, M., Tarakhovsky, A., and Rajewsky, K. (2008). BAFF activates
Akt and Erk through BAFF-R in an IKK1-dependent manner in primary mouse
B cells. Proc. Natl. Acad. Sci. USA 105, 12435–12438.
Patke, A., Mecklenbra ¨uker, I., Erdjument-Bromage, H., Tempst, P., and
Tarakhovsky, A. (2006). BAFF controls B cell metabolic fitness through a
PKC beta- and Akt-dependent mechanism. J. Exp. Med. 203, 2551–2562.
Rahman, Z.S., and Manser, T. (2004). B cells expressing Bcl-2 and asignaling-
impaired BAFF-specific receptor fail to mature and are deficient in the forma-
tion of lymphoid follicles and germinal centers. J. Immunol. 173, 6179–6188.
Rahman, Z.S., Rao, S.P., Kalled, S.L., and Manser, T. (2003). Normal induction
but attenuated progression of germinal center responsesin BAFF and BAFF-R
signaling-deficient mice. J. Exp. Med. 198, 1157–1169.
Ramadani, F., Bolland, D.J., Garcon, F., Emery, J.L., Vanhaesebroeck, B.,
Corcoran, A.E., and Okkenhaug, K. (2010). The PI3K isoforms p110alpha
and p110delta are essential for pre-B cell receptor signaling and B cell
development. Sci. Signal. 3, ra60.
Razani, B., Zarnegar, B., Ytterberg, A.J., Shiba, T., Dempsey, P.W., Ware,
C.F., Loo, J.A., and Cheng, G. (2010). Negative feedback in noncanonical
NF-kappaB signaling modulates NIK stability through IKKalpha-mediated
phosphorylation. Sci. Signal. 3, ra41.
Rickert, R.C., Rajewsky, K., and Roes, J. (1995). Impairment of T-cell-
dependent B-cell responses and B-1 cell development in CD19-deficient
mice. Nature 376, 352–355.
Rickert, R.C., Jellusova, J., and Miletic, A.V. (2011). Signaling by the tumor
necrosis factor receptor superfamily in B-cell biology and disease. Immunol.
Rev. 244, 115–133.
Rowland, S.L., Leahy, K.F., Halverson, R., Torres, R.M., and Pelanda, R.
(2010). BAFF receptor signaling aids the differentiation of immature B cells
into transitional B cells following tonic BCR signaling. J. Immunol. 185,
Sasaki, Y., Casola, S., Kutok, J.L., Rajewsky, K., and Schmidt-Supprian, M.
(2004).TNF familymemberBcell-activating factor(BAFF)receptor-dependent
and -independent roles for BAFF in B cell physiology. J. Immunol. 173, 2245–
Sasaki, Y., Derudder, E., Hobeika, E., Pelanda, R., Reth, M., Rajewsky, K., and
Schmidt-Supprian, M. (2006). Canonical NF-kappaB activity, dispensable for
B cell development, replaces BAFF-receptor signals and promotes B cell
proliferation upon activation. Immunity 24, 729–739.
Sasaki, Y., Calado, D.P., Derudder, E., Zhang, B., Shimizu, Y., Mackay, F.,
Nishikawa, S.-i., Rajewsky, K., and Schmidt-Supprian, M. (2008). NIK overex-
pression amplifies, whereas ablation of its TRAF3-binding domain replaces
BAFF:BAFF-R-mediated survival signals in B cells. Proc. Natl. Acad. Sci.
USA 105, 10883–10888.
Schiemann, B., Gommerman, J.L., Vora, K., Cachero, T.G., Shulga-Morskaya,
S., Dobles, M., Frew, E., and Scott, M.L. (2001). An essential role for BAFF in
the normal development of B cells through a BCMA-independent pathway.
Science 293, 2111–2114.
Schweighoffer, E., Vanes, L., Nys, J., Cantrell, D., McCleary, S., Smithers, N.,
and Tybulewicz, V.L. (2013). The BAFF receptor transduces survival signals by
co-opting the B cell receptor signaling pathway. Immunity 38, 475–488.
Senftleben, U., Cao, Y., Xiao, G., Greten, F.R., Kra ¨hn, G., Bonizzi, G., Chen,Y.,
Hu, Y., Fong, A., Sun, S.C., and Karin, M. (2001). Activation by IKKalpha of a
second, evolutionary conserved, NF-kappa B signaling pathway. Science
Shinners, N.P., Carlesso, G., Castro, I., Hoek, K.L., Corn, R.A., Woodland,
R.T., Scott, M.L., Wang, D., and Khan, W.N. (2007). Bruton’s tyrosine kinase
mediates NF-kappa B activation and B cell survival by B cell-activating factor
receptor of the TNF-R family. J. Immunol. 179, 3872–3880.
Smith, S.H., and Cancro, M.P. (2003). Cutting edge: B cell receptor signals
regulate BLyS receptor levels in mature B cells and their immediate progeni-
tors. J. Immunol. 170, 5820–5823.
So, L., and Fruman, D.A. (2012). PI3K signalling in B- and T-lymphocytes: new
developments and therapeutic advances. Biochem. J. 442, 465–481.
Srinivas, S., Watanabe, T., Lin, C.-S., William, C.M., Tanabe, Y., Jessell, T.M.,
and Costantini, F. (2001). Cre reporter strains produced by targeted insertion
of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4.
Stadanlick, J.E.,Kaileh, M., Karnell, F.G., Scholz, J.L., Miller, J.P., Quinn, W.J.,
3rd, Brezski, R.J., Treml, L.S.,Jordan, K.A., Monroe, J.G.,et al.(2008). Tonic B
1034 Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors
cell antigen receptor signals supply an NF-kappaB substrate for prosurvival Download full-text
BLyS signaling. Nat. Immunol. 9, 1379–1387.
Suzuki, A., Kaisho, T., Ohishi, M., Tsukio-Yamaguchi, M., Tsubata, T., Koni,
P.A., Sasaki, T., Mak, T.W., and Nakano, T. (2003). Critical roles of Pten in B
cell homeostasis and immunoglobulin class switch recombination. J. Exp.
Med. 197, 657–667.
Tardivel, A., Tinel, A., Lens, S., Steiner, Q.G., Sauberli, E., Wilson, A., Mackay,
F., Rolink, A.G., Beermann, F., Tschopp, J., and Schneider, P. (2004). The
anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival
but not for the marginal zone B cell differentiation activity of BAFF. Eur. J.
Immunol. 34, 509–518.
Thompson, J.S., Bixler, S.A., Qian, F., Vora, K., Scott, M.L., Cachero, T.G.,
Hession, C., Schneider, P., Sizing, I.D., Mullen, C., et al. (2001). BAFF-R, a
newly identified TNF receptor that specifically interacts with BAFF. Science
Vallabhapurapu, S., Matsuzawa, A., Zhang, W., Tseng, P.-H., Keats, J.J.,
Wang, H., Vignali, D.A.A., Bergsagel, P.L., and Karin, M. (2008). Nonredundant
and complementary functions of TRAF2 and TRAF3 in a ubiquitination
cascade that activates NIK-dependent alternative NF-kappaB signaling. Nat.
Immunol. 9, 1364–1370.
Vikstrom, I., Carotta, S., Lu ¨thje, K., Peperzak, V., Jost, P.J., Glaser, S.,
Busslinger, M., Bouillet, P., Strasser, A., Nutt, S.L., and Tarlinton, D.M.
(2010). Mcl-1 is essential for germinal center formation and B cell memory.
Science 330, 1095–1099.
Vora, K.A., Wang, L.C., Rao, S.P., Liu, Z.Y., Majeau, G.R., Cutler, A.H., Hoch-
man, P.S., Scott, M.L., and Kalled, S.L. (2003). Cutting edge: germinal centers
formed in the absence of B cell-activating factor belonging to the TNF family
exhibit impaired maturation and function. J. Immunol. 171, 547–551.
Woodland, R.T., Fox, C.J., Schmidt, M.R., Hammerman, P.S., Opferman, J.T.,
Korsmeyer, S.J., Hilbert, D.M., and Thompson, C.B. (2008). Multiple signaling
pathways promote B lymphocyte stimulator dependent B-cell growth and
survival. Blood 111, 750–760.
Xiao, G., Harhaj, E.W., and Sun, S.C. (2001). NF-kappaB-inducing kinase
regulates the processing of NF-kappaB2 p100. Mol. Cell 7, 401–409.
Xie, P., Stunz, L.L., Larison, K.D., Yang, B., and Bishop, G.A. (2007). Tumor
necrosis factor receptor-associated factor 3 is a critical regulator of B cell
homeostasis in secondary lymphoid organs. Immunity 27, 253–267.
Zarnegar,B.,Yamazaki, S., He, J.Q., and Cheng, G.(2008a). Control of canon-
ical NF-kappaB activation through the NIK-IKK complex pathway. Proc. Natl.
Acad. Sci. USA 105, 3503–3508.
Zarnegar, B.J., Wang, Y., Mahoney, D.J., Dempsey, P.W., Cheung, H.H., He,
J., Shiba, T., Yang, X., Yeh, W.C., Mak, T.W., et al. (2008b). Noncanonical
NF-kappaB activation requires coordinated assembly of a regulatory complex
of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK. Nat.
Immunol. 9, 1371–1378.
3-kinase represses IgE switch by potentiating BCL6 expression. J. Immunol.
Cell Reports 5, 1022–1035, November 27, 2013 ª2013 The Authors 1035